Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes.

Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets.

Combinatorial plasma polymerization approach to produce thin films for testing cell proliferation.

Plasma enhanced physical vapor depositions are extensively used to fabricate substrates for cell culture applications. One peculiarity of the plasma processes is the possibility to deposit thin films with reproducible chemical and physical properties. In the present work, a combinatorial plasma polymerization process was used to deposit thin carbon based films to promote cell adhesion, in the interest of testing cell proliferation as a function of the substrate chemical properties. Peculiarity of the combinatorial approach is the possibility to produce in just one deposition experiment, a set of surfaces of varying chemical moieties by changing the precursor composition. A full characterization of the chemical, physical and thermodynamic properties was performed for each set of the synthesized surfaces. X-ray photoelectron spectroscopy was used to measure the concentration of carboxyl, hydroxyl and amine functional groups on the substrate surfaces. A perfect linear trend between polar groups' density and precursors' concentration was found. Further analyses reveled that also contact angles and the correspondent surface energies of all deposited thin films are linearly dependent on the precursor concentration. To test the influence of the surface composition on the cell adhesion and proliferation, two cancer cell lines were utilized. The cell viability was assessed after 24 h and 48 h of cell culture. Experiments show that we are able to control the cell adhesion and proliferation by properly changing the thin film deposition conditions i.e. the concentration and the kind of chemical moiety on the substrate surface. The results also highlight that physical and chemical factors of biomaterial surface, including surface hydrophobicity and free energy, chemical composition, and topography, can altered cell attachment.

Efectos de un programa multiprofesional de tratamiento de la obesidad sobre los factores de riesgo para síndrome metabólico en niños prepúberes, púberes y adolescentes: diferencias entre géneros / Effects of a multidisciplinary program of obesity treatment on risk factors for metabolic syndrome in children in prepubertal, pubertal and adolescent stages: differences between genders

Objetivo. Analizar los efectos de un programa multidisciplinar de tratamiento de la obesidad (PMTO) sobre los factores de riesgo del Síndrome Metabólico (SM) en niños prepúberes, púberes y adolescentes de acuerdo con el género. Método. Participaron en el estudio 69 niños y adolescentes obesos entre 10 y 18 años de edad divididos en dos grupos: grupo de intervención (GI) (n = 37) y grupo control (GC) (n = 32). En el GI había 23 niñas, en el GC 14. El GI fue sometido a intervención multidisciplinar, con duración de 16 semanas. Se evaluaron parámetros antropométricos, aptitud cardiorrespiratoria y factores de riesgo para SM. Resultados. Se observó que el GI obtuvo reducción en la prevalencia de SM (- 35,8 % para género masculino y - 8,7 % para femenino), entre tanto se mantuvo el valor en las niñas del grupo GC y aumentó en los niños del GC (+ 11,1 %). En relación a dislipidemias, hubo una reducción en el GI para ambos géneros (- 7,2 % para el masculino; - 17,4 % para el femenino), y para el GC se observó aumento para el masculino (+ 22,2 %) y femenino (14,3 %). Las niñas del GI tuvieron mejoras significativas para las variables índice de masa corporal, circunferencia de cintura y cadera, y sensibilidad a la insulina, que no fueron observadas en el género masculino del GI, que presentaron aumento de masa magra. Conclusión. Los resultados del estudio muestran que 16 semanas de intervención multidisciplinar, basada en una terapia cognitivo-conductual, son suficientes para promover reducción de la prevalencia de SM y dislipidemias en niños y adolescentes obesos (AU)

Objetive. To analyze the effects of a multidisciplinary program of obesity treatment (PMTO) on risk factors for metabolic syndrome (MS) in children and adolescents at prepubertal, pubertal and adolescents stages according to gender. Method. he study included 69 obese children and adolescents aged 10 to 18 years. They were allocated to the intervention group (GI) (n = 37) and control group (GC) (n = 32). There were 23 girls in GI and 14 in GC. The GI was submitted to the multidisciplinary intervention, lasting 16 weeks. It was assessed anthropometric parameters, cardiorespiratory fitness and risk factor for MS. Results. We found that the GI achieved a reduction in the prevalence of MS (7.1 % for boys and 8.7 % for girls), and in GC a maintenance for girls and increasing for boys. For dyslipidemia, a reduction in GI for both genders (boys 78.6 % to 71.4 %; girls 82.6 % to 65.2 %), and increasing in GC for both genders. GI Girls had significant improvements for the variables, body mass index, waist circumference, hip circumference and insulin sensibility which was not observed in GI boys who increased lean body mass. Conclusion. The findings show that 16 weeks of multidisciplinary intervention based on cognitive behavioral therapy are sufficient to promote decreasing in the prevalence of MS and dyslipidemia in obese children and adolescents (AU)

Multifunctional branched gold-carbon nanotube hybrid for cell imaging and drug delivery.

Branched gold nanoparticles were grown on oxidized multiwalled carbon nanotubes by one-step reduction of gold chloride in water. The carbon nanotube/gold hybrids were used for the delivery of the anticancer drug doxorubicin hydrochloride into A549 lung cancer cell line. Doxorubicin (Dox) can be adsorbed in high quantity on both inner and outer surfaces of oxidized carbon nanotubes by π-π stacking interactions between doxorubicin aromatic groups and carbon nanotube (CNT) backbone. Carbon nanotube/gold hybrids display a broad absorption band in the red and near-infrared regions allowing their use for imaging applications. In vitro cellular tests showed that the nanostructures can efficiently transport and deliver doxorubicin inside the cells.

Sustained in vitro release and cell uptake of doxorubicin adsorbed onto gold nanoparticles and covered by a polyelectrolyte complex layer.

Gold nanoparticles functionalized with doxorubicin and stabilized with multilayers of degradable polyelectrolyte were allowed to age in aqueous medium in vitro in order to show the possibility of drug release in cellular environment. The chemico-physical characteristics of the nanoparticles are reported. The observed release of doxorubicin (DOX) was pH-dependent, and it increased in acidic environment. Cell uptake of nanoparticles and drug release were monitored by laser scanning confocal microscopy. Data showed that drug-bearing nanoparticles delivered DOX into the nuclei of A549 cells, leading to pronounced cytotoxic effects to this lung tumor cells. Our results suggest that gold nanoparticles conjugated with doxorubicin could be used as a pH-triggered drug releasing carrier for tumor drug delivery.

Non-invasive pressure support ventilation in acute hypoxemic (non hypercapnic) respiratory failure. Observations in Respiratory Intermediate Intensive Care Unit.

BACKGROUND: Non-invasive positive pressure support ventilation (NIPSV). METHODS: In patients with acute hypoxaemic (PaO2/FiO2 &Mac178;100) non hypercapnic respiratory failure (ARF) admitted to a Respiratory Inter-mediate Intensive Care Unit of a general Hospital, between January 1993 and December 1997. RESULTS: In 21 selected patients (PaO2/ FiO2T0=82+/-9) NIPSV improved PaO2 in 13/21 patients (Group A) and did not improve in 8/21 patients (Group B) (PaO2/FiO2T1=154+/-25 in Group A vs PaO2/FiO2T1=106+/-7.5 in Group B, p=0.00001). Upon admission the two groups did neither significantly differ for blood gas values (PaO2/FiO2T0=84+/-9.6 in Group A vs 79.8+/-8.7 in Group B), nor for clinical status (APACHE II=19.8+/-5 in Group A vs 24.6+/-7 in Group B). Shorter duration of NIPSV in Group B patients (11.2+/-19.7 hrs vs 35.3+/-32.3 hrs in Group A, p=0.047), in spite of a rise in PEEP (9.3+/-2.3 in Group B vs 5.5+/-2.4 in Group A, p=0.003) and Pressure Support (18.7+/-1.8 in Group B vs 15+/-3.2 in Group A, p=0.004) was due to onset of conditions which required shifting from NIPSV to endotracheal intubation (ETI). OUTCOME: 8/21 patients were successfully treated by only NIPSV. 8/21 patients were intubated. 5/21 patients dead in RIICU; 1 month survival: 9/21 patients. Side effects: mask intolerance (3/21); skin necrosis (1/21); pneumothorax (1/21). CONCLUSIONS: NIPSV may be tried in ARF patients to improve PaO2 and avoid ETI.

The role of erythrocytapheresis in secondary erythrocytosis therapy.

In chronic respiratory insufficiency secondary erythrocytosis (SPC), causing pulmonary hypertension and dx ventricular insufficiency, is often noticed. An alternative therapy to phlebotomy for SPC is isovolemic large volume erythrocytapheresis performed with cell separator (CSE) in order to quickly remove a large volume of red blood cells (RBC) while saving plasma proteins and clotting factors. In order to evaluate the efficiency and safety of CSE in SPC we reported a retrospective analysis of our experience with 61 SPC patients: from April 1996 to May 1998 we performed 208 CSE using Haemonetics MCS3P (TAE protocol). Before every apheresis procedure we verified Hb (in median 18.8 g/dl), Ht (in median 58.4%), viscometry, coagulation test, EGA, PFR and ECG. 11 patients were treated with 1 CSE, 12 with 3, 29 with 4 and 9 with 5. The mean volume of RBC removed was 576 ml (range 426-800); Hb post CSE averaged 14.4 g/dl and Ht post CSE averaged 42.7%; hematic viscosity post CSE was significantly reduced while tissue oxygen tension increased: the improvement of symptomatology and hematochemical parameters was maintained on the average for 6.5 months. All the procedures were well tolerated and light side effects (paresthesias citrate-depending in 27 apheresis) were easily controlled. CSE, compared to phlebotomy, has the advantage of selectively removing RBC without loss of clotting factors, platelets and plasma proteins. Although CSE has relatively high costs we noticed a decrease of hospital recurrence (about 50-65%) in SPC patients treated with apheresis.

Transient ischemic attack in a patient with congenital protein-C deficiency during treatment with stanozolol.

A patient with congenital protein-C deficiency was treated with stanozolol for 8 weeks to increase circulating levels of protein C. A rise in protein C was achieved, accompanied by an increase in factor II, factor X, antithrombin III, and protein S; but at the 8th week the patient suffered a transient ischemia attack.