Priming with a very low dose of DNA complexed with cationic block copolymers followed by protein boost elicits broad and long-lasting antigen-specific humoral and cellular responses in mice

Cationic block copolymers spontaneously assemble via electrostatic interactions with DNA molecules in aqueous solution giving rise to micellar structures that protect the DNA from enzymatic degradation both in vitro and in vivo. In addition, we have previously shown that they are safe, not immunogenic and greatly increased antigen-specific CTL responses following six intramuscular inoculations of a very low dose (1 μg) of the vaccine DNA as compared to naked DNA. Nevertheless, they failed to elicit detectable humoral responses against the antigen. To gain further insight in the potential application of this technology, here we show that a shorter immunization protocol based on two DNA intramuscular inoculations of 1 μg of DNA delivered by these copolymers and a protein boost elicits in mice broad (both humoral and cellular) and long-lasting responses and increases the antigen-specific Th1-type T cell responses and CTLs as compared to priming with naked DNA. These results indicate that cationic block copolymers represent a promising adjuvant and delivery technology for DNA vaccination strategies aimed at combating intracellular pathogens.     

Screen sensitivity in photosensitive children and adolescents: patient-dependant and stimulus-dependant factors.

Television viewing is the most frequent cause of photogenic attacks in daily life. In the present study, we examined 48 photosensitive children and adolescents to find out: 1) whether hypersynchronous activity is induced less often by viewing a PC monitor than a television screen and 2) whether certain images are more likely to cause hypersynchronous activity than others. All subjects were tested for sensitivity to intermittent photic stimulation (IPS) and to a black and white striped pattern on cards. Additionally, all were subjected to stimuli from four different images (vertical black and white striped pattern, geometric figures, text, and a painting by Max Pechstein - 1913, Italian church), presented on a television screen (with an image regeneration frequency of 50 Hz) and on PC screens (with regeneration frequencies of 48 and 100 Hz). A total of 21 non-photosensitive, healthy children and adolescents served as controls. Of the 48 photosensitive subjects 13% were also pattern sensitive (cards), and 33% exhibited screen sensitivity. No differences were found between the three monitor types. However, the hypersynchronous reactivity to the four images presented was significantly different, with high contrast vertical striped pattern being the most provocative. Non-photosensitive subjects did not react to any of the stimuli. The results of the present study show that screen-dependant factors are less important than image-dependant factors.     

Angiotensin-(1–7) stimulates water transport in rat inner medullary collecting duct: evidence for involvement of vasopressin V2 receptors

The peptide angiotensin-(1–7) [Ang-(1–7)] is known to enhance water transport in rat inner medullary collecting duct (IMCD). The aim of this study was to determine the mechanism of the Ang-(1–7) effect on osmotic water permeability (P _f). P _f was measured in the normal rat IMCD perfused in vitro in presence of agonists [Ang-(1–7), arginine vasopressin (AVP) and Ang-(3–8)], and antagonists of the angiotensin and the vasopressin cascade. Ang-(1–7), but not Ang-(3–8), increased P _f significantly. The effect of Ang-(1–7) on P _f was abolished by its selective antagonist, A-779, added before or after Ang-(1–7). Prostaglandin E₂ and the protein kinase A inhibitor H8 also blocked the Ang-(1–7) effect. Blockade of vasopressin V₁ receptors by antagonists did not change the Ang-(1–7) effect, but pre-treatment with a V₂ antagonist abolished the effect of Ang-(1–7) on P _f. Similarly, pre-treatment with A-779 inhibited AVPs effect on P _f. Forskolin-stimulated P _f was blocked both by A-779 and by the V₂ antagonist. Finally, Ang-(1–7) increased cAMP levels in fresh IMCD cell suspensions whilst the forskolin-stimulated cAMP synthesis was decreased by A-779 and the V₂ antagonist. These data provide evidence that Ang-(1–7) interacts via its receptor with the AVP V₂ system through a mechanism involving adenylate-cyclase activation.     

Histopathological parameters and lymphatic metastasis in supraglottic laryngeal carcinoma]

Histopathologic parameters in predicting lymph node metastasis of supraglottic laryngeal carcinoma. A systematic clinical-pathological study was performed on fifty-three resected cases of supraglottic laryngeal squamous cell carcinoma, followed up for at least 5 years. The aim of the research was to evaluate histopathologic parameters in predicting lymph node metastasis (N+) as expression of biological malignancy of the neoplasm. The following neoplastic microscopical features were studied: histopathologic and cytologic grading, pattern of growth, peritumoral inflammatory infiltrate, stromal reaction, tumoral necrosis. The results are as follows: stromal reaction and cytologic grading are not useful to identify N+ and N- cases. Cases with high and low degree of differentiation (Broder's grading) are significantly correlated respectively to low (14.3%) and high (70%) incidence of lymph node metastasis (p less than 0.03). A clear correlation is present between the pattern of growth "pushing" and lacking of node metastasis (84.6%). A favorable prognosis significance seems to be linked with the presence of peritumoral lymphoplasmocytic infiltrate, which results to be a marker of cases in which lymph node metastasis incidence is very low (5.5%; p less than 0.001). On the contrary lymph node metastasis incidence increase when tumoral necrosis is present (76.5%; p less than 0.001).     

Protection of killer antiidiotypic antibodies against early invasive aspergillosis in a murine model of allogeneic T-cell-depleted bone marrow transplantation

Antiidiotypic monoclonal antibodies (MAbs) representing the internal image of a yeast killer toxin (KT) have therapeutic potential against several fungal infections. The efficacy of KT MAbs against Aspergillus fumigatus was investigated in a mouse model of T-cell-depleted allogeneic bone marrow transplantation (BMT) with invasive pulmonary aspergillosis. Mice were highly susceptible to infection at 3 days post-BMT, when profound neutropenia was observed both in the periphery and in the lungs. Treatment with KT MAbs protected the mice from infection, as judged by the long-term survival and decreased pathology associated with inhibition of fungal growth and hyphal development in the lungs. In vitro, similar to polymorphonuclear neutrophils, KT MAbs significantly inhibited the hyphal development and metabolic activity of germinated Aspergillus conidia. These results indicate that mimicking the action of neutrophils could be a strategy through which KT MAbs exert therapeutic efficacy in A. fumigatus infections.     

Interleukin-7-engineered mesenchymal cells: in vitro effects on naive T-cell population.

T-cell homeostasis is regulated by several molecules; among these, interleukin (IL)-7 plays an essential role in the survival and homeostatic proliferation of peripheral naive T cells. In a previous study, we investigated whether human mesenchymal stromal cells (MSCs) could be engineered with the IL-7 gene to produce functional level of this cytokine. In the present study, we analyzed the impact of different quantities of IL-7 produced by MSCs on the survival and proliferation of a negative immunoselected naive (CD3(+)/CD45RA(+)) T-cell population. Co-cultivation of peripheral naive T cells with MSCs producing low (16 pg/mL) or high (1000 pg/mL) IL-7 levels or in the presence of exogenous IL-7 (0.01 ng/mL and 100 ng/mL) maintained the CD3(+)/CD45RA(+) naive T-cell phenotype. Chemokine receptor CCR7(+) expression was also maintained among this T-cell population. Naive T-cell molecular characteristics were maintained as assessed by the Vbeta spectratyping complexity score, which showed the maintenance of a broad T-cell repertoire. No Th1 or Th2 differentiation was observed, as assessed by interferon-gamma or IL-4 accumulation. In contrast, only MSCs producing high amounts of IL-7 caused increased activation (CD25 31.2% +/- 12% vs 10% +/- 3.5%; P < .05), proliferation (CD71 17.8+/-7% vs 9.3%+/-3, P < .05), apoptosis (assessed by annexin V: 18.6% +/- 5% vs 14.9% +/- 2.6%; P > .05), and the phase S cell cycle (15% vs 6.9%, P > .05). Exogenous IL-7 exhibited no significant effect. In conclusion, we demonstrated that IL-7 produced by MSCs has a dose-independent effect on naive T-cell survival while exerting a dose-dependent effect on activation/proliferation. Due to the continuous production of IL-7 by engineered cells, our system is more efficacious than exogenous IL-7.