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.     

Modulation of Ca2+ levels in keratinocytes by all-trans retinoic acid.

Human epidermal keratinocytes, that have been growth-arrested by removal of epidermal growth factor from the culture medium, are stimulated to proliferate by all-trans retinoic acid (RA). The same treatment inhibits the onset of differentiated features and reduces cell-substrate adhesion. In the present study we show that the same treatment results in a decrease in total cell-associated Ca2+ as measured by changes in the amount of 45Ca2+ bound to cells at equilibrium following RA treatment and by a decrease in intracellular free Ca2+ levels as measured with the Ca(2+)-sensitive dye, Indo-1. The alterations in Ca2+ levels were evident within an hour after RA treatment, were in the range of 30-35% and occurred over the same RA concentration range that stimulated proliferation (i.e., 0.25-1.0 micrograms/ml). When the extracellular Ca2+ concentration was elevated from the normal level of 0.15-1.4 mM, intracellular free Ca2+ increased by a factor of 2 while total cell-associated Ca2+ increased approximately 6-fold. Even under conditions of high extracellular Ca2+, RA was able to reduce cell-associated and intracellular free Ca2+. These data indicate that RA has the capacity to lower Ca2+ levels in keratinocytes concomitantly with its effects on biological behavior.     

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.     

Peripheral hemodynamic and humoral effects of oral zofenopril calcium (SQ. 26,991) in patients with congestive heart failure

In 14 patients with congestive heart failure (CHF) of various grade (NYHA class 2-4) the effects of zofenopril calcium (SQ 26,991) on blood pressure and forearm circulation were studied by venous occlusion plethysmography. Changes in plasma renin activity (PRA), aldosterone, Atrial natriuretic factor (ANF) and arginine-vasopressin (AVP) were also measured. Two hours after oral administration of 7.5 mg of zofenopril we observed a decrease in blood pressure, heart rate, and forearm vascular resistance along with an increase in venous distensibility. Zofenopril also decreased ANP levels in a manner directly related to peripheral venodilatation (r = .64; P less than .05) and modified arginine-vasopressin (AVP) proportionally to the fall in blood pressure observed in response to drug administration (%SBP/%AVP: r = .64, P less than .05; %DBP/%AVP: r = .67, P less than .05). Hemodynamic and humoral responses to zofenopril occurred without any significant unwanted adverse reaction, even in patients with greater pressor reduction. We conclude that oral acute zofenopril administration, in patients with congestive heart failure, causes an arterial and venous forearm vasodilatation which is probably involved in the acute changes in plasma levels of ANF and AVP observed after drug administration.     

Beta-adrenoceptor subtypes in young and old rat ventricular myocytes: a combined patch-clamp and binding study.

1. We used electrophysiological and binding techniques to assess the presence of beta 1- and beta 2-adrenoceptors (beta 1AR and beta 2AR) in rat cardiac myocytes and to determine their ratio during aging. Experiments were performed in left ventricular myocytes enzymatically dissociated from the heart of 3-(young) or 22-month-old (old) Wistar Kyoto rats. 2. In patch-clamp experiments, myocytes from old rats showed a prolonged action potential duration (at -20 mV: 41.7 +/- 3.6 vs 26.2 +/- 3.1 ms; at -60 mV: 154.4 +/- 17.7 vs 87.1 +/- 6.9 ms, P < 0.05) and an augmented membrane capacitance (an index of cell size) (271.7 +/- 20.2 vs 164.3 +/- 14.6 pF, P < 0.05) compared to young rats. beta 2AR stimulation, achieved by superfusing myocytes with the selective beta 2AR agonist, zinterol (10 microM) or with (-)-isoprenaline (1 microM) in the presence of the selective beta 1AR antagonist, CGP 20712A (0.1 microM), significantly increased L-type calcium current (ICa,L) in rat ventricular myocytes. The percentage increase was similar in both young and old rats, either with zinterol (26.9 +/- 3.6% and 24.2 +/- 2.8%, respectively) or isoprenaline plus CGP 20712A (30.4 +/- 3.7% and 22.4 +/- 4.1%, respectively). Isoprenaline alone (beta 1AR and beta 2AR stimulation) caused a much smaller increase in ICa,L in old rats (58.4 +/- 12.1%) than in younger ones (95.3 +/- 8.1%) (P = 0.067).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Maternal IgG avidity and IgM detected by blot as diagnostic tools to identify pregnant women at risk of transmitting cytomegalovirus

In this study, we determined the avidity index (AI) of anticytomegalovirus (CMV) immunoglobulin G (IgG) and the anti-CMV immunoglobulin M (IgM) profile in 124 pregnant women, 87 of whom were considered at risk of transmitting CMV infection to their offspring and 37 of whom were at no risk. IgG avidity and blot for IgM were performed on two serum samples from each woman, at 6-18 weeks' gestation and at 20-23 weeks' gestation. Pregnancy outcomes were monitored. The results obtained showed that the determination of anti-CMV IgG avidity at 6-18 weeks' gestation can identify all women who would have an infected fetus/newborn (100% sensitivity), whereas IgM detected by blot had poorer results (69% sensitivity). Interestingly, at 20-23 weeks' gestation, the sensitivity of IgM detection by blot was higher than that obtained by avidity (75 % and 63%, respectively) and the combination of IgG avidity and IgM by blot yielded the best results (81% sensitivity).