Influence of surface coating on the intracellular behaviour of gold nanoparticles: a fluorescence correlation spectroscopy study.

In the biomedical applications of nanoparticles (NPs), the proper choice of surface chemistry is a crucial aspect in their design. The nature of the coating can heavily impact the interaction of NPs with biomolecules, affect the state of aggregation, and ultimately determine their biological fate. As such, protein corona formation and the aggregation behaviour of gold NPs (Au NPs) are studied here. Au NPs are prepared with four distinct surface functionalisations, namely mercaptosuccinic acid (MSA), N-4-thiobutyroil glucosamine, HS-PEG5000 and HS-alkyl-PEG600. Corona formation, aggregation, and the intracellular behaviour of the Au NPs are then investigated by means of Fluorescence Correlation Spectroscopy (FCS) in cell culture media and in live cells. To evaluate the state of aggregation and the formation of a protein corona, the Au NPs are incubated in cell media and the diffusion coefficient is determined via FCS. The in vitro behaviour is compared with the level of aggregation of the NPs in cells. Diffusion times of the NPs are estimated at different positions in the cell after a one hour incubation period. It is found that the majority of MSA and glucose-Au NPs are present inside the cell as slowly diffusing species with diffusion times (τD) greater than 6000 µs (hydrodynamic diameter >250 nm). PEGylated Au NPs adsorb a small amount of protein and manifest low agglomeration both in media and in living cells. In particular, the HS-alkyl-PEG600 coating shows an excellent correlation between lower protein adsorption, 4-fold lower compared to the MSA coated NPs, and limited intracellular aggregation. In the case of single HS-alkyl-PEG600 coated NPs, it is found that typical intracellular τD values range from 500 to 1500 µs, indicating that these particles display reduced aggregation in the intracellular environment.

Hepatitis E virus infection in Latin America: a review.

Data reported during recent years reveal the complex picture of the epidemiology of hepatitis E virus (HEV) infection in Latin America. Whereas in countries like Argentina and Brazil is almost identical to the characteristic of most countries from North America and Europe, HEV in the Caribbean and Mexico involves the water-borne, non-zoonotic viral genotypes responsible for epidemics in Asia and Africa. Nevertheless, Latin America has been considered a highly endemic region for hepatitis E in the scientific literature, a generalization that ignores the above complexity. In addition, reports from isolated Amerindian communities, which display well known, important and very specific epidemiological features for hepatitis B and D virus infections are neither taken into account when considering the epidemiology of hepatitis E in the region. This review updates compilation of the available information for the HEV infection, both among humans and other mammals, in Latin America, discusses the strengths and the weaknesses of our current knowledge, and identifies future areas of research.

Seroprevalencia de marcadores de hepatitis B y factores de riesgo asociados en pacientes de hemodiálisis de Ciudad de La Habana; 2002-2003 / Prevalence of hepatitis B virus markers and risk factors associated in haemodialysis patients from Havana city; 2002-2003

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Efficacy of antiviral agents in feline herpetic keratitis: results of an in vitro study.

PURPOSE: To determine, by a plaque reduction assay, the in vitro efficacy of novel antiviral agents in the treatment of feline herpes virus 1 (FHV-1) keratitis in the domestic cat (Felis felis). MATERIALS AND METHODS: A standard plaque reduction assay was performed using a laboratory strain of FHV-1 and embryo-derived feline kidney cells to determine the in vitro efficacy of the antiviral drugs penciclovir (PCV), bromovinyldeoxyuridine (BVdU), and (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) and to compare these with the drugs acyclovir (ACV) and trifluorothymidine (TFT). Efficacy was assessed by determining the dose of drug at which 50% plaque reduction was noted (ED(50)). RESULTS: HPMPA was found to have greatest antiviral activity (ED(50) 0.07 microg/ml). ACV was least active (ED(50) 24 microg/ml), while TFT was active with an ED(50) of 5.7 microg/ml. PCV and BVdU had intermediate activity (ED(50) 1.6 and 1.7 microg/ml, respectively). CONCLUSIONS: This study suggests that the efficacy of HPMPA, BVdU, and penciclovir in cats with herpesviral keratitis should be determined in vivo as their efficacy in vitro was substantially greater than that of acyclovir, already shown to have demonstrable but limited clinical antiviral activity.

Visual signals in an optomotor reflex: systems and information theoretic analysis.

Compensatory optomotor reflexes were examined in crayfish (Procambarus clarkii) with oscillating sine wave gratings and step displacements of a single stripe. A capacitance transducer was used to measure the rotation of the eyestalk about its longitudinal axis. System studies reveal a spatial frequency response independent of velocity and stimulus amplitude and linear contrast sensitivity similar to that of neurons in the visual pathway. The reflex operates at low temporal frequencies (<0.002 Hz to 0.5 Hz) and exhibits a low-pass temporal frequency response with cut-off frequency of 0.1 Hz. Eyestalk rotation increases as a saturable function of the angular stimulus displacement. When compared to the oscillatory response, transient responses are faster, and they exhibit a lower gain for large stimulus displacements. These differences may reflect system nonlinearity and/or the presence of at least two classes of afferents in the visual pathway. Our metric for information transmission is the Kullback-Leibler (K-L) distance, which is inversely proportional to the probability of an error in distinguishing two stimuli. K-L distances are related to differences in responsiveness for variations in spatial frequency, contrast, and angular displacement. The results are interpreted in terms of the neural filters that shape the system response and the constraints that the K-L distances place on information transmission in the afferent visual pathway.

Pharmacological analysis of cannabinoid receptor activity in the rat vas deferens.

1. The interaction between the cannabinoid agonists, WIN 55,212-2 or CP 55,940 with the CB(1) receptor-selective antagonists, SR141716A or LY320135 was investigated using the rat electrically-stimulated vas deferens bioassay. 2. Tissues were stimulated by single-field pulses (150 V, 0.5 ms) delivered every 30 mins. In the presence of nifedipine (3 microM), agonists elicited a concentration-dependent inhibition of the contractile response, with pEC(50) values of 7.93 and 6.84 for WIN 55,212-2 and CP 55,940, respectively. 3. SR141716A and LY320135 caused parallel dextral displacements of the agonist concentration-response curves. However, the shift of the agonist curves by either antagonist was accompanied by a concentration-dependent enhancement of basal (agonist-independent) tissue contraction. 4. Addition of the amidase inhibitor, phenylmethylsulphonylfluoride (200 microM), resulted in a significant reduction of the basal twitch response, an effect consistent with the presence of tonic receptor activation mediated by the endogenous cannabinoid, anandamide. 5. In light of these findings, we propose a theoretical model of competitive agonist-antagonist interaction in the presence of endogenous agonist tone that was used to derive an optimized analytical approach for the determination of antagonist potency estimates under conditions of tonic receptor activation. 6. This approach yielded pK(B) estimates for SR141716A and LY320135 that were in good agreement with their activity at cannabinoid CB(1) receptors. 7. It is concluded that the rat vas deferens contains prejunctional cannabinoid CB(1) receptors that are under tonic activation from endogenous substances; under these conditions our analytical approach is preferable to the standard methods for the determination of antagonist potency.

Effect of amlodipine on cardiopulmonary performance in volunteers.

1. In order to exclude a significant effect of the calcium channel antagonist amlodipine on cardiopulmonary performance in normal subjects, we performed a double-blind cross-over study of amlodipine (10 mg daily for 2 weeks) on oxygen uptake and catecholamine responses during exercise in eight volunteers. 2. Despite a therapeutic plasma concentration of amlodipine (22.8+/-9 ng/mL), there was no change in resting heart rate or blood pressure. Amlodipine did not cause significant change in oxygen uptake at the anaerobic threshold or at maximum exercise and there was also no change in heart rate or catecholamine responses. 3. Although there was an awareness of peripheral vasodilation and reports of lethargy during the active treatment period, the volunteers had no objective evidence of a decrease in cardiopulmonary performance. We suggest that use of amlodipine as a vasodilator in the perioperative period would not add to the myocardial depressant effects of general anaesthesia in patients with normal cardiac function.

Persistencia de anti-HBs en ancianos inmunizados con una vacuna ADN recombinante anti-hepatitis B: dos años de seguimiento / Anti-HBs persistent to immune aged with a DNA recombinant anti-hepatitis B vaccine: two years of follow

Se aplicó la vacuna recombinante cubana contra el virus de la hepatitis B Herberbiovac HB a un grupo de individuos mayores de 65 años, pertenecientes a un hogar de ancianos de Ciudad de la Habana, a la dosis de 20 ug, con el esquema 0,1 y 6 meses para estudiar su inmunogenicidad, la cual fue evaluada a los 2, 7, 12 meses y 2 años después de aplicada la primera dosis. A los 60 días se obtuvo un 47,1 por ciento de títulos protectores (>10 UI/L) y media geométrica (TMG) de 6,04 UI/L. Cuando se evaluó la respuesta inmune a los 7 meses se logró un 100,0 por ciento de seroprotección (>10 UI/L); 78,6 por ciento de buenos respondedores (títulos antiHBs >100 UI/L) y media geométrica de 136,93 UI/L. Al año, la seroprotección también fue del 100 por ciento, y se logró 61,5 por ciento de títulos >100 UI/L y la media geométrica de 204,61 UI/L. La persistencia de niveles de anticuerpos protectores y TMG 2 años después de aplicada la primera dosis de la vacuna fue de 71,4 por ciento y 24,73 UI/L, respectivamente. Estos resultados evidencian el alto poder inmunogénico de esta vacuna, lo que la hace recomendable para la protección de este grupo de edad contra el virus de la hepatitis B

Pharmacological characterisation of cannabinoid CB(1) receptors in the rat and mouse.

The role of cannabinoid CB(1) receptors in sympathetic neurotransmission was characterised in nerve-mediated responses of isolated right atria, vasa deferentia and small mesenteric resistance arteries using the cannabinoid CB(1) receptor agonists Delta(9)-tetrahydrocannabinol, CP 55,940 and anandamide and the cannabinoid CB(1)-selective antagonist SR 141716A. In the mouse vas deferens, the twitch response was completely inhibited by each of the putative cannabinoid receptor agonists with pIC(50) values of CP 55,940, 9.2+/-0.1; Delta(9)-tetrahydrocannabinol, 8.4+/-0.1; anandamide, 7.1+/-0.1. SR 141716A 10-100 nM was a competitive antagonist of all three agonists with a pK(B) value of 8.4-8.6, consistent with an interaction at the cannabinoid CB(1) receptor. In the rat vas deferens CP 55,940 (0.01-10 microM) inhibited the contractions to a significant extent (88.5+/-0.5% at 10 microM; pIC(50) of 7.1+/-0.1) while Delta(9)-tetrahydrocannabinol and anandamide (both up to 10 microM) were inactive. CP 55,940 exhibited low potency in rat compared with mouse vas deferens and the rat concentration-response curve was not competitively antagonised by SR 141716A (100 nM) or SR 144528 (10 nM-10 microM), suggesting an interaction at a receptor(s) distinct from cannabinoid CB(1) or CB(2). Sympathetic nerve-induced tachycardia in rat and mouse atria, and rat mesenteric artery smooth muscle contractile responses to perivascular nerve stimulation, were not inhibited by Delta(9)-tetrahydrocannabinol, CP 55,940 or anandamide up to 1 microM. These data indicate that cannabinoid CB(1) receptor activation inhibits sympathetic neurotransmission only in the mouse vas deferens and thus point to species and regional differences in cannabinoid CB(1) receptor involvement in pre-synaptic inhibition of sympathetic neurotransmission and CP 55,940 may have inhibitory actions in rat vas deferens unrelated to cannabinoid receptor activity.

[Immunogenicity of the Heberbiovac-HB vaccine in doses of 10.5 and 2.5 micrograms in 6-9-year-old students]. / Inmunogenicidad de la vacuna Heberbiovac-HB a las dosis de 10,5 y 2,5 micrograms en niños escolares de 6 a 9 años de edad.

The Heberbiovac-HB recombinant hepatitis B vaccine was administered at dosages of 10.5 and 2.5 micrograms in 3 groups of children aged 6-9 with a vaccination schedule at 0, 1 and 6 months of age. The immunogenecity attained between dosages was compared. The seroconversion with protective titres obtained at 2 months varied from 88.9% with 10 micrograms to 79.4% with 2.5 micrograms, whereas at 7 months it was higher than 98% in the 3 groups. The geometrical mean with 10 mg was 136.63 and 2,356.8 UI/L-1; with 5 micrograms, 77.62 and 1,958.9 UI/L-1; and with 2.5 micrograms, 32.15 and 376.3 UI/UL-1, at 2 and 7 months, respectively. A high immunogenic power of the vaccine is observed at dosages of 10 and 5 micrograms with similar results. In spite of the fact that the dosage of 2.5 micrograms is immunogenic, the response is not desirable. These results state the possibility of using a lower dosage (5 micrograms) for a low risk infantile population, which favors the cost-benefit.

[Infection/immunity by hepatitis B virus in an adult population of Havana City. Cuba 1995]. / Infección/inmunidad por el virus de la hepatitis B en una población adulta de Ciudad de La Habana. Cuba 1995.

The results of a study of markers of infection/immunity by hepatitis B virus in a community of the health area in "Cristobal Labra" polyclinics, City of Habana in 1995. The studied healthy adult population was not included in any vaccination strategy with hepatitis B vaccine. Two markers were studied: Hepatitis B surface antigen and its corresponding antibody (anti-HBs). HBsAg-positivity was 1.05% and anti-HBs positivity was 13.39%. Of anti-HBs-positive cases, 13.39% had titers from 10-50 UI/L and the rest (43.68%) had titers over 50 UI/L.

[Filter paper sampling for the detection of anti-hepatitis A IgM antibodies]. / Toma de muestra en papel de filtro para la detección de anticuerpos IgM antivirus de la hepatitis A.

The usefulness of the whole sample taking in filter paper for the detection of anti-HAV IgM is described. 168 serum and filter paper samples were simultaneously compared. No alterations were observed in the specificity of the enzyme-linked immunosorbent assay for the catchment of IgM in the 52 samples of the control group or in the other 116 from patients with clinical suspicion of acute viral hepatitis. A sensitivity, specificity and coincidence of 100% was obtained on comparing the filter paper with the dilution 1:500 with the serum. The correlation established between the values of optical density of the positive results to anti-HAV IgM, the filter paper at dilution 1:500 and the serum was of 0.895. On applying this type of sample taking in a group of hospitalized patients, 100% of coindicence with clinical suspicion was attained. Besides, it was proved its usefulness for the diagnosis of a viral hepatitis outbreak. According to our results, it is recommended the introduction of this type of sample taking in the diagnosis of hepatitis A.

Synthesis of N-acetylglucosamine containing Lewis A and Lewis X building blocks based on N-tetrachlorophthaloyl protection--synthesis of Lewis X pentasaccharide.

Phenyl 6-O-benzyl-2-deoxy-2-tetrachlorophthalimido-1-thio-beta-D- glucopyranoside (5a) and thexyldimethylsilyl 6-O-benzyl-2-deoxy-2-tetrachlorophthalimido-beta-D- glucopyranoside (5b) gave with O-(2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl)trichloroacetimida te (8) in the presence of BF3.Et2O as catalyst exclusively lactosamine derivatives 7a and 7b, respectively, in high yields. Ensuing reaction with O-(3, 4-di-O-acetyl-2-O-benzyl-alpha-L-fucopyranosyl) trichloroacetimidate (9) in the presence of TMSOTf as catalyst afforded Le(x) trisaccharide intermediates 10a,b. With fucosyl donor 9 and 5a,b as acceptors in the presence of TMSOTf as catalyst glycosylation either at the 3-O or the 4-O was observed, thus leading to mixtures of disaccharides 11a/12a and 11b/12b, respectively; their reaction with 8 furnished Le(x) trisaccharide intermediates 10a,b and Le(a) trisaccharide intermediates 14a,b. Transformation of 10b into the corresponding trichloroacetimidate 17 and reaction with lactose acceptor 19 in the presence of Zn(OTf)2 as catalyst gave protected Le(x) pentasaccahride intermediate 21, which on deprotection led to unprotected Le(x) pentasaccharide 1.

Capsular polysaccharide of Streptococcus pneumoniae type 19F: synthesis of the repeating unit.

A new and more versatile synthesis of beta-D-ManpNAc-(1-->4)-alpha-D-Glcp-(1-->2)-alpha-L-Rhap, the trisaccharide repeating unit of the Streptococcus pneumoniae type 19F capsular polysaccharide, is described. The present approach allows a simple access to different fragments containing the trisaccharide and the conjugation of the product(s) to a protein through the selective manipulation of the anomeric position at the reducing end and of the HO-4 function at the nonreducing end. The synthetic scheme shows an efficient application of the sulfoxide method for the stereoselective and high yielding formation of the glycosidic linkages.

A simple access to lactose-derived building blocks required in glycoconjugate synthesis.

Lactose was readily transformed into thexyldimethylsilyl (3,4-O-isopropylidene-beta-D-galactopyranosyl)-(1-->4)-beta- D-glucopyranoside (5); this compound served as intermediate for the generation of partially O-protected lactose building blocks required in oligosaccharide and glycoconjugate synthesis. Thus, from 5 via per-O-benzoylation, desilylation, trichloroacetimidate formation, glycosylation of the Lemieux spacer, and acid-catalyzed de-O-isopropylidenation methoxycarbonyloctyl (2,6-di-O-benzoyl-beta-D-galactopyranosyl)- (1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranoside (12) was obtained. Regioselective benzoylation of 5 with benzoyl cyanide under various conditions afforded 3-O- (13), 2,3,2'-O- (14), 3,2'-O- (16), and 2,2'-O-unprotected (17) lactoside, respectively. De-O-isopropylidenation of 16 gave thexyldimethylsilyl (6-O-benzoyl-beta-D-galactopyranosyl)-(1-->4)-2, 6-di-O-benzoyl-beta-D-glucopyranoside (18), an important 2',3',4'-O-unprotected lactose derivative. Fucosylation of 13 and then de-O-isopropylidenation afforded thexyldimethylsilyl 2,6-di-O-benzoyl-beta-D-galactopyranosyl)-(1-->4)-[(3,4-di-O-acetyl-2-O- benzoyl-alpha-L-fucopyranosyl)-(1-->3)]-2,6-di-O-benzoyl-beta-D- glucopyranoside (21), an important fucosyllactose building block.

Adenoviral vectors efficiently target cell lines derived from selected lymphocytic malignancies, including anaplastic large cell lymphoma and Hodgkin's disease.

We have hypothesized that adenoviral vectors might mediate gene transfer into cell lines derived from human lymphocytic malignancies, such as lymphoma, lymphocytic leukemia, and myeloma. A panel of 33 cell lines was studied for their ability to be transduced by an adenoviral (AD) vector encoding the Escherichia coli beta-galactosidase gene (AD-betagal). A cytochemical assay and a flow cytometry assay both demonstrated that a subset of lymphocytic cell lines can be efficiently transduced by adenoviral vectors. In particular, three of three anaplastic large cell lymphoma lines, two of two Hodgkin's disease cell lines, two of seven Burkitt's lymphoma cell lines, and three of five myeloma cell lines exhibited efficient gene transfer. The ability of an AD vector expressing the thymidine kinase (tk) gene from herpes simplex virus-1 (AD-tk) followed by ganciclovir (GCV) to kill 11 of these lymphocytic cell lines was studied. In eight of the cell lines tested, more than 68% of the cells were killed by AD-tk/GCV. Similar results were obtained using an adenoviral vector expressing the wild-type p53 tumor suppressor gene (AD-p53). Thus, AD-tk/GCV and AD-p53 both demonstrated efficient killing of these cell lines. These data document that adenoviral vectors are valuable reagents for the introduction of genes into selected lymphocytic cell lines. These data also suggest that adenoviral vectors might be useful for gene therapy of subsets of lymphocytic malignancy.

[Immunogenicity of the Heberbiovac-HB vaccine in the physically and mentally handicapped]. / Inmunogenicidad de la vacuna Heberbiovac-HB en impedidos físicos y mentales.

The recombinant DNA Heberbiovac-HB vaccine against hepatitis B was applied to 2 groups of children at 2 homes for the physically and mental disabled at doses of 10 and 5 micrograms with the scheme 0, 1 and 6 months to study its immunogenicity, which was evaluated at 2, 7 and 12 months after the first dose was administered. On the 60th day it was obtained 80.9% of seroprotection in children who received 10 micrograms, whereas in the group that was vaccinated with 5 micrograms 65.0% of seroprotection was attained and there were no significant differences between the groups. 100% of seroprotection (> or = 10 UI/L) was obtained in the 2 vaccinated groups when the immune response was measured 1 month after the third dose was applied. A year later, seroprotection was 100% for the 2 groups of children. The geometrical mean of the antibody titres (TMG) reached levels above 10 UI/L, which is the minimum protector, in the 2 study groups 30 days after the second dose. A month after the third dose, the TMG attained values of 527.7 UI/L in the group of 10 micrograms and of 324.7 UI/L in that of 5 micrograms. No remarkable differences were found between the groups (p < 0.05). Finally, 365 days later, the TMG descended to 139.7 UI/L and 57.3 UI/L in the same groups, respectively. It was demonstrated the high immunogenic power of this vaccine, which makes it recommendable for protecting the physical and mentally disabled children from hepatitis B virus.