Fixation patterns evaluation by means of MP-1 microperimeter in microstrabismic children treated for unilateral amblyopia.

PURPOSE: The aim of the study was to evaluate the fixation patterns of microstrabismic children previously treated for unilateral amblyopia. METHODS: Thirty-three children (mean age 7.3+/-1.5 years) were included in the study. Visual acuity (VA) was measured using the Early Treatment of Diabetic Retinopathy Study charts. Fixationwas assessed by MP-1 microperimeter. Differences in position and stability of fixation between the fellow and the microstrabismic eyes were calculated by using the percentage of the preferred fixation points within central fixation and the percentage of the fixation points within target fixation, respectively. For statistical analysis Mann-Whitney test was used. To evaluate the influence of age and duration of anti-amblyopic treatment on microstrabismic eyes fixation, linear regression analysis was performed. RESULTS: In the microstrabismic eyes VA was significantly reduced when compared to the fellow eyes (0.1236+/-0.0204 vs 0.0042+/-0.0032 logMAR; p<0.001). Position and stability of fixation were significantly better in the fellow eyes (93.21+/-0.65% vs 70.91+/-4.80%; p=0.002, and 89.88+/-0.94% vs 71.73+/-2.94%; p<0.001, respectively). A significant correlation was found between fixation stability and both the duration of anti-amblyopic treatment and pretreatment VA (p=0.024 and p=0.009, respectively) and between fixation centrality and pretreatment VA (p<0.001). CONCLUSIONS: VA, centrality, and stability of fixation were significantly impaired in the microstrabismic eyes. Pretreatment VA was a risk factor for fixation impairment. The severity of fixation stability impairment was linked to the duration of anti-amblyopic treatment.

A sensitive cell-based assay for the detection of residual infectious West Nile virus.

Ensuring complete viral inactivation is critical for the safety of vaccines based on an inactivated virus. Detection of residual infectious virus is dependent on sensitivity of the assay, sample volume analyzed and the absence of interference with viral infection. Here we describe the development and qualification of a sensitive cell-based assay for the detection of residual infectious West Nile Virus (WNV). The results of the assay are in good agreement with the assumption that at low concentrations the number of infectious units in relatively small samples follows a Poisson distribution. The assay can detect 1 infectious unit with a confidence of 99%, provides statistical controls for interference and can easily be scaled up to test large amounts of vaccine material. Furthermore, we show equivalence in sensitivity between the cell-based assay and an in vivo assay for detection of infectious WNV. Finally, the assay has been used for successful release testing of clinical lots of inactivated WNV vaccine. Given the principle and generic setup of the method we envision broad applicability to the detection of very low concentrations of infectious virus.

A replication-competent adenovirus assay for E1-deleted Ad35 vectors produced in PER.C6 cells.

The presence of replication-competent adenovirus (RCA) is a safety concern for biologics based on recombinant adenoviruses and RCA testing is therefore mandatory for release of clinical material. RCA, which arises from homologous recombination between Ad5 vectors and HEK-293 cells, can be eliminated by the use of PER.C6 cells in combination with a matched vector. However, little is known on RCA formation with vectors based on adenovirus serotypes other than Ad5 and reliable RCA assays to test them are generally lacking. Here we report on the development and qualification of a sensitive RCA assay for Ad35, a promising alternative to Ad5 vectors. The assay is able to detect 1 RCA in 3x10(10) vector particles with 95% confidence, thus meeting current FDA requirements, and can discriminate between RCA and other rare CPE-causing entities, including helper dependent E1 positive particles (HDEP). Using this assay, the first batches of Ad35 vectors produced in PER.C6 cells were analysed and found to be free of RCA and HDEP. Based on the statistical model used, we anticipate that our approach to RCA assay development can be broadly applicable to other adenoviral vectors.

A rapid method for immunotitration of influenza viruses using flow cytometry.

Reliable assays for accurate titration of influenza virus in infectious samples are pivotal to both influenza research and vaccine development. A titration assay adopted commonly for this purpose is the plaque assay on Madin-Darby canine kidney (MDCK) cells, despite it being time and labour consuming. A novel assay is described for titration of influenza viruses based on the detection of intracellular viral nucleoprotein (NP) by fluorescence-activated cell sorting (FACS). By using a panel of viruses of different type, subtype and origin, it is demonstrated that there is a mathematical correlation between titres measured by immunotitration and by classical plaque assay on MDCK cells. Moreover, the availability of NP antibodies specific for type A or type B influenza virus ensures the specificity of the assay. Based on speed, accuracy and specificity, it is concluded that the FACS-based immunotitration of influenza virus represents a valid and efficient alternative to the classical plaque assay.

In vitro evolution of a highly replicating, doxycycline-dependent HIV for applications in vaccine studies.

A major concern associated with the use of vaccines based on live-attenuated viruses is the possible and well documented reversion to pathogenic phenotypes. In the case of HIV, genomic deletions or mutations introduced to attenuate viral pathogenicity can be repaired by selection of compensating mutations. These events lead to increased virus replication rates and, eventually, disease progression. Because replication competence and degree of protection appear to be directly correlated, further attenuation of a vaccine virus may compromise the ability to elicit a protective immune response. Here, we describe an approach toward a safe attenuated HIV vaccine. The system is not based on permanent reduction of infectivity by alteration of important viral genomic sequences, but on strict control of replication through the insertion of the tetracycline (Tet) system in the HIV genome. Furthermore, extensive in vitro evolution was applied to the prototype Tet-controlled HIV to select for variants with optimized rather than diminished replication capacity. The final product of evolution has properties uniquely suited for use as a vaccine strain. The evolved virus is highly infectious, as opposed to a canonically attenuated virus. It replicates efficiently in T cell lines and in activated and unstimulated peripheral blood mononuclear cells. Most importantly, replication is strictly dependent on the nontoxic Tetanalogue doxycycline and can be turned on and off. These results suggest that this in vitro evolved, doxycycline-dependent HIV might represent a useful tool toward the development of a safer, live-attenuated HIV vaccine.

Strict control of human immunodeficiency virus type 1 replication by a genetic switch: Tet for Tat.

Live-attenuated human immunodeficiency virus type 1 (HIV-1) variants have shown great promise as AIDS vaccines, but continued replication can lead to the selection of faster-replicating variants that are pathogenic. We therefore designed HIV-1 genomes that replicate exclusively upon addition of the nontoxic effector doxycycline (dox). This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression. These designer "HIV-rtTA" viruses replicate in a strictly dox-dependent manner both in a T-cell line and in primary blood cells, and the rate of replication can be fine-tuned by simple variation of the dox concentration. These HIV-rtTA viruses provide a tool to perform genetics, e.g., selection and optimization experiments, with the E. coli-derived Tet reagents in a eukaryotic background. Furthermore, such viruses may represent improved vaccine candidates because their replication can be turned on and off at will.

E2F family members are differentially regulated by reversible acetylation.

The six members of the E2F family of transcription factors play a key role in the control of cell cycle progression by regulating the expression of genes involved in DNA replication and cell proliferation. E2F-1, -2, and -3 belong to a structural and functional subfamily distinct from those of the other E2F family members. Here we report that E2F-1, -2, and -3, but not E2F-4, -5, and -6, associate with and are acetylated by p300 and cAMP-response element-binding protein acetyltransferases. Acetylation occurs at three conserved lysine residues located at the N-terminal boundary of their DNA binding domains. Acetylation of E2F-1 in vitro and in vivo markedly increases its binding affinity for a consensus E2F DNA-binding site, which is paralleled by enhanced transactivation of an E2F-responsive promoter. Acetylation of E2F-1 can be reversed by histone deacetylase-1, indicating that reversible acetylation is a mechanism for regulation also of non-histone proteins.

Chromatin control of HIV-1 gene expression.

Upon infection of susceptible cells, the RNA genome of the human immunodeficiency virus type 1 (HIV-1) is reverse transcribed into double-stranded DNA, which can be subsequently integrated into the cellular genome. After integration, the viral long terminal repeat (LTR) promoter is present in a nucleosome-bound conformation and is transcriptionally silent in the absence of stimulation. Activation of HIV-1 gene expression is concomitant with an acetylation-dependent rearrangement of the nucleosome positioned at the viral transcription start site. Thus, similar to most cellular genes, the transcriptional state of the integrated HIV-1 provirus is closely linked to histone acetylation. This enzymatic activity results from the function of histone-specific nuclear acetyltransferase (HAT) enzymes. Efficient viral transcription is strongly dependent on the virally-encoded Tat protein. the mechanism by which Tat increases the rate of transcriptional initiation has been recently demonstrated and involves the interaction of Tat with the transcriptional coactivator p300 and the closely related CREB-binding protein (CBP), having histone acetyltransferase activity.

HIV-1 tat transactivator recruits p300 and CREB-binding protein histone acetyltransferases to the viral promoter.

In cells infected with HIV type 1 (HIV-1), the integrated viral promoter is present in a chromatin-bound conformation and is transcriptionally silent in the absence of stimulation. The HIV-1 Tat protein binds to a stem-loop structure at the 5' end of viral mRNA and relieves this inhibition by inducing a remodeling of the nucleosome arrangement downstream of the transcription-initiation site. Here we show that Tat performs this activity by recruiting to the viral long terminal repeat (LTR) the transcriptional coactivator p300 and the closely related CREB-binding protein (CBP), having histone acetyltransferase (HAT) activity. Tat associates with HAT activity in human nuclear extracts and binds to p300 and CBP both in vitro and in vivo. Integrity of the basic domain of Tat is essential for this interaction. By a quantitative chromatin immunoprecipitation assay we show that the delivery of recombinant Tat induces the association of p300 and CBP with the chromosomally integrated LTR promoter. Expression of human p300 in both human and rodent cells increases the levels of Tat transactivation of the integrated LTR. These results reinforce the evidence that p300 and CBP have a pivotal function at both cellular and viral promoters and demonstrate that they also can be recruited by an RNA-targeted activator. Additionally, these findings have important implications for the understanding of the mechanisms of HIV-1 latency and reactivation.

Quantitative dynamics of HIV type 1 expression.

A competitive PCR and RT-PCR procedure was developed for the quantification of HIV-1 nucleic acids in infected biological samples, with particular reference to the study of the kinetics of production of differently processed viral transcripts. The procedure entails the utilization of a competitor plasmid DNA (on DNA samples) or of an in vitro transcription product obtained from this plasmid (on RNA samples) and allows the quantification of proviral DNA, viral genomic RNA, and viral single- and multispliced mRNAs. Furthermore, it permits the direct standardization of these measurements to the amount of a reference cellular gene (for DNA quantification) or of a reference cellular transcript (for RNA quantification). This quantification procedure was used to monitor the dynamics of HIV-1 transcriptional activation in the latently infected U1 monocytic cell line after stimulation with phorbol-12-myristate-13-acetate, and in experimentally infected peripheral blood lymphocytes. Despite the biological differences between the two experimental systems, in both cases production of infectious virus is accompanied by a remarkable increase in the levels of unspliced viral mRNAs (rising up to 20,000 fold in U1 cells) and by a consequent switch in the abundance of the differently spliced transcript classes. These observations reinforce the notion that the control of infection is subjected also to posttranscriptional events and prompts for quantitative evaluation of HIV-1 transcript class abundance in infected individuals to define potential markers for disease progression.

Molecular and functional interactions of transcription factor USF with the long terminal repeat of human immunodeficiency virus type 1.

The human transcription factor USF, purified from HeLa cells, and its recombinant 43-kDa component bind to the long terminal repeat (LTR) of human immunodeficiency virus type 1. The proteins footprint over nucleotides from position -173 to -157 upstream of the transcription start site, generating strong DNAse I hypersensitivity sites at the 3' sides on both strands. As detected by methylation protection studies, the factor forms symmetric contacts with the guanines of the palindromic CACGTG core of the recognized sequence. Its binding ability is abolished by the mutation of this core sequence and is strongly reduced by the cytosine methylation of the central CpG dinucleotide. Upon binding, both recombinant and purified USFs bend the LTR DNA template. The role of USF in the control of transcription initiation from the LTR was tested by in vitro transcription assays. Upon addition of the protein, transcription from constructs containing an intact binding site is increased, while the responsiveness in constructs with a mutated sequence is abolished. Furthermore, the addition of a decoy plasmid which contains multiple repeats of the target sequence results in downregulation of transcription from the LTR. These results suggest that USF is a positive regulator of LTR-mediated transcriptional activation.

[Analysis of prognostic factors in 106 cases of carcinoma of the rhinopharynx treated with cobalt teletherapy]. / Analisi dei fattori prognostici in 106 casi di carcinoma della rinofaringe trattati con telecobaltoterapia.

A series of 106 patients affected with nasopharyngeal carcinomas and treated by definitive external irradiation from January 1975 to December 1986 was retrospectively reviewed. The median follow-up, from the end of the treatment, was 43 months (range 24-90). The nasopharynx received not less than 60 Gy to the midplane: the clinically negative neck (N0) was treated with a total dose of 50 Gy and the patients who had N1-3 disease received not less than 60 Gy. Thirty-eight patients had a recurrence in the irradiated areas (31 in the nasopharynx, and 7 in the neck); 17 patients developed distant metastases. Disease-free survival at 60 months was 42%. The most significant prognostic factor (p less than 0.05) was the presence of advanced neck involvement (N2-3), since most of the lymphatic and distant recurrences were observed in this group of patients. The overall results did not reveal but slight differences in the survival according to histology, even though patients with undifferentiated carcinomas had a local recurrence rate significantly lower than those with squamous cell carcinomas. Our findings suggest that patients with N2-3 neck diseases or with locally advanced involvement (T3-4) be treated by adjuvant chemotherapy in order to decrease the risk of local and distant relapses.