Use of a short-term whole blood intracellular staining assay to study the T-cell response in respiratory syncytial virus-infected pediatric patients.

The aim of the present study was the development of a reliable method to evaluate the pattern of the ongoing T-cell response in young infants affected by respiratory infection. To this purpose, we enrolled 44 infants hospitalized with a diagnosis of respiratory syncytial virus bronchiolitis. After a short-term stimulation of whole blood samples, intracellular IFN-g and IL-4 cytokines were measured in CD4+ and CD8+ T-cell subsets by flow cytometry. A stringent staining and gating strategy was used in order to maximize the reduction of background noise and to exclude false positives. The frequencies of cytokine-producing T-cell subsets, albeit low, were easily quantifiable. Cytokine responses were higher in infants sampled > 7 days from the onset of symptoms. The use of a rigorous strategy for cell staining and gating, coupled with a short-term stimulation of whole blood and a careful evaluation of time elapsed from the onset of symptoms constitutes a convincing approach for future clinical studies.

Infants hospitalized for Bordetella pertussis infection commonly have respiratory viral coinfections.

BACKGROUND: Whether viral coinfections cause more severe disease than Bordetella pertussis (B. pertussis) alone remains unclear. We compared clinical disease severity and sought clinical and demographic differences between infants with B. pertussis infection alone and those with respiratory viral coinfections. We also analyzed how respiratory infections were distributed during the 2 years study. METHODS: We enrolled 53 infants with pertussis younger than 180 days (median age 58 days, range 17­109 days, 64. 1% boys), hospitalized in the Pediatric Departments at "Sapienza" University Rome and Bambino Gesù Children's Hospital from August 2012 to November 2014. We tested in naso-pharyngeal washings B. pertussis and 14 respiratory viruses with real-time reverse-transcriptase-polymerase chain reaction. Clinical data were obtained from hospital records and demographic characteristics collected using a structured questionnaire. RESULTS: 28/53 infants had B. pertussis alone and 25 viral coinfection: 10 human rhinovirus (9 alone and 1 in coinfection with parainfluenza virus), 3 human coronavirus, 2 respiratory syncytial virus. No differences were observed in clinical disease severity between infants with B. pertussis infection alone and those with coinfections. Infants with B. pertussis alone were younger than infants with coinfections, and less often breastfeed at admission. CONCLUSIONS: In this descriptive study, no associations between clinical severity and pertussis with or without co-infections were found. TRIAL REGISTRATION: Policlinico Umberto I: protocol 213/14, 3085/13.02.2014, retrospectively registered. Bambino Gesù Children's Hospital: protocol n. RF-2010-2317709.

The HIV-1 Nef protein: how an AIDS pathogenetic factor turns to a tool for combating AIDS.

Nef is one of the six regulatory proteins coded by the Human Immunodeficiency Virus (HIV)-1 and -2, and by the Simian Immunodeficiency Virus (SIV). Accumulating experimental evidences indicate that Nef is required for the optimal infectivity of HIV viral particles, and that it plays a critical role in the AIDS pathogenesis progressing. We previously cloned and sequenced a functionally defective HIV-1 genome (F12HIV-1) whose nef gene showed a rather unusual feature, i.e. its expression blocks the HIV-1 release by interfering with the viral assembling/release. Such a striking phenotype appeared to be the result of three amino acid substitutions, and coupled with the loss of the most part of the Nef functions described for the wild type counterpart. The F12Nef properties encouraged the designing of new strategies of anti HIV-1 gene therapy we afforded by recovering an inducible lentivirus vector expressing F12Nef as the cytoplasmic domain of a transmembrane fusion protein including a selectable marker (i.e. the Nerve Growth Factor receptor) as the ecto- and transmembrane domains. As expected, the expression of such a chimeric protein resulted in a potent protection of transduced cells from the HIV-1 spread. In sum, and surprisingly enough, we generated a reagent effectively counteracting the HIV-1 replication through the combination of a slightly mutated AIDS pathogenetic factor together with a lentivirus vector, i.e. the result of artifactual modifications of the HIV-1 genome.

Oligomerization of RAR and AML1 transcription factors as a novel mechanism of oncogenic activation.

RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.