Probing insulin's secondary structure after entrapment into alginate/chitosan nanoparticles.

The aim of the present study was to probe the structural integrity of insulin after being entrapped into chitosan/alginate nanoparticles produced by ionotropic polyelectrolyte pre-gelation. By manipulating the alginate:chitosan mass ratio and the pH during nanoparticle production, desired nanoparticles with a mean size of 850 (+/-88)nm and insulin association efficiency of 81 (+/-2)% were obtained. Insulin secondary structure was assessed by Fourier transform infrared (FTIR) and circular dichroism (CD) after entrapment into nanoparticles and after release from the particles under gastrointestinal simulated conditions. FTIR second-derivative spectra and area-overlap compared to an insulin standard confirmed that no significant conformational changes of insulin occurred in terms of alpha-helix and beta-sheet content. Far-UV-CD spectra corroborated the preservation of insulin structure during the nanoparticle production procedure. The presented nanoparticulate system is a promising carrier for insulin oral delivery since it preserves insulin structure and therefore also, potentially, its bioactivity.     

Therapy of post-renal transplantation hyperlipidaemia: comparative study with simvastatin and fish oil

Background: Recipients of renal transplantation (RT) exhibit disturbances of serum lipids and apoproteins that may contribute to their cardiovascular morbidity and mortality. In our renal transplant department the hypercholesterolaemia prevalence at the first and fifth year of RT is 70.0% and 81.2%, respectively. Lipid-lowering therapy has been utilized in many Transplant Units. The aim of our study was to evaluate post-RT hyperlipidaemia control with simvastatin or fish oil. Method: Forty-three RT patients (26 men and 17 women) with persistent hypercholesterolaemia and stable graft function which were resistant to a lipid-lowering diet (American Heart Association Step Two) were randomized into two groups and treated for 3 months with simvastatin (S) (10 mg/day; n=25) and fish oil (F) (6 g/day; n=18). Total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), lipoprotein a (Lp(a)), apolipoprotein A1 (Apo A1), and apolipoprotein B (Apo B) were monitored and at the study baseline they were similar between the two groups. Results: No side effects were detected after 3 months of therapy. In group S, the concentrations of TC (271±46 mg% vs 228±49mg%; P <0.001), TG (180±78 vs 134±45; P<0.01), LDL-C (177& plusmn;40 vs 144±43; P <0.01) and Apo B (96±18 vs 82±16; P <0.001) were significantly reduced, and Apo A1 concentration had increased (135±24 vs 149±30; P <0.01). In group F, the concentrations of TC (266±25 vs 240±31; P <0.001), TG (203±105 vs 156±72; P=0.02) and HDL-C (63±15 vs 53±12; P <0.01) were significantly reduced. Conclusion: We concluded that low-dose simvastatin and fish oil are both effective and safe in correcting post-RT hyperlipidaemia. Further prospective studies with larger follow-up are needed to clarify whether this therapy has an impact on cardiovascular morbidity and mortality in RT patients.     

Involvement of reactive oxygen intermediates in tumor necrosis factor alpha-dependent bacteriostasis of Mycobacterium avium.

We studied the involvement of reactive oxygen intermediates and reactive nitrogen intermediates in the bacteriostasis of two Mycobacterium avium strains differing in virulence by resident peritoneal macrophages. We found that both the highly virulent strain (25291) and the low-virulence strain (1983) of M. avium induced superoxide production but inhibited nitrite production in vitro. This inhibition was due to the production of superoxide, a nitric oxide scavenger. The stimulation of superoxide production was two- to fivefold higher in strain 1983-infected than in strain 25291-infected resident peritoneal macrophages and was independent of contaminating T cells or NK cells. Superoxide secretion was dependent on the tumor necrosis factor (TNF) produced endogenously by the macrophages. This was also true when macrophages were isolated from infected mice. Addition of TNF to the infected resident peritoneal macrophages caused only a slight, albeit significant, increase in superoxide production by strain 25291-infected macrophages. Incubation of resident peritoneal macrophages with different scavengers of reactive oxygen intermediates showed that strain 1983 was susceptible to hydrogen peroxide produced by resident peritoneal macrophages. Strain 25291 was shown to decrease superoxide secretion inside heavily infected bone marrow-derived macrophages. This strain was also shown to be a better trigger for production of reactive oxygen intermediates than strain 1983. In summary, strain 1983 induced high levels of TNF synthesis that acted in an autocrine fashion to stimulate production of reactive oxygen intermediates by macrophages leading to growth restriction mediated by hydrogen peroxide. The highly virulent strain 25291 induced low levels of TNF synthesis, and therefore little reactive oxygen intermediate production, and could also inhibit superoxide production by the infected macrophages.     

Abnormal extrapelvic course of the inferior gluteal artery

At its extrapelvic course the inferior gluteal a. is found to be strictly related to the sciatic n. This relationship has been described in a general way, emphasizing its medial localization in respect to the nerve. Clinicosurgical reports describe cases of aneurysms of the inferior gluteal a. on its extrapelvic course and subsequent compression at the nerve. In order to get further details on the relationship between these two structures, 80 gluteal regions from 40 cadavers of adult Brazilian individuals, 29 males and 11 females, were dissected. The inferior gluteal a. was found medial to the sciatic n. in 62 cases (77.5%); in the 18 remaining (22.5%) the trunk of the artery or one of its branches perforated the nerve. Of these, 14 (77.8%) were males and 4 (22.2%) females. This disposition was found 8 times (44.4%) on the right and 10 (55.6%) on the left side; was unilateral in 4 individuals (1 on the right and 3 on the left side) and bilateral in 7. The course of the inferior gluteal a. through the sciatic n. and/or the presence of aneurysms of this artery should be considered as a possible cause of nerve compression.     

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF): identification of a binding site for a neutralizing antibody.

One approach to the localization of functionally active regions of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is to map the epitopes recognized by neutralizing anti-hGM-CSF monoclonal antibodies. We have defined the epitope recognized by one neutralizing antibody (LMM102) using proteolytic fragments obtained by enzymic digestion of bacterially synthesized hGM-CSF. RP-HPLC fractionation of a tryptic digest resulted in the identification of an immunoreactive "tryptic core" peptide containing 66 amino acids (52% of the protein). Further digestion of this "tryptic core" with S. aureus V8 protease produced a unique immunoreactive hGM-CSF product comprising two peptides, residues 86-93 and 112-127, linked by a disulfide bond between residues 88 and 121. The individual peptides, generated by reduction with dithiothreitol, were not recognized by the antibody. An analog of this peptide has been synthesized chemically and shown to have similar immunoreactivity to the epitope obtained by enzymic digestion. A series of modified peptides has also been synthesized to identify further the region required for antibody recognition.     

Self-selection in responding to a health risk appraisal: are we preaching to the choir?

Abstract The objectives of the present study were a) to examine behavioral and sociodemographic factors associated with voluntary response to a health risk appraisal (HRA), and b) to assess the effect of HRA feedback on subsequent preventive health behaviors and risk-taking behaviors. After collection of health behavior data from a larger sample participating in a Navy-wide health promotion evaluation, an HRA was mailed to a random subsample of 625 individuals. A total of 270 (43 %) people responded to the HRA and received printed feedback. Follow-up health behavior data were collected one year later from 93 of the 270 HRA respondents. These 93 individuals were then matched with a control group who did not receive an HRA but had provided health behavior data both times. Analyses revealed that HRA respondents were older, better educated, had higher health status, smoked less, consumed less alcohol, and used seat belts more often than nonrespondents. Separate analyses of HRA respondents and matched controls indicated that HRA participation had no significant effect on subsequent preventive health behaviors or risk-taking behaviors. Results suggest limitations of the HRA as an effective due to behavior change.     

Dynamics of experimental vasogenic brain oedema in the rat: changes induced by adrenergic drugs

The effects of adrenergic drugs on the formation and resolution of cerebral oedema in a rat model of cold-induced vasogenic brain oedema were studied. Evans blue dye extravasation, water content and ultrastructural alterations (pinocytotic vesicle formation in capillary endothelial cells and apparent water accumulation in the brain parenchyma) were evaluated in parietal cortex. Previous administration of the alpha-adrenoceptor antagonist phenoxybenzamine produced a reduction of Evans blue extravasation and water content, diminished vesicle formation and reduced water accumulation. Previous administration of the beta2-adrenoceptor agonist clenbuterol reduced Evans blue extravasation and water content, but did not change vesicle frequency. The effects of clenbuterol on Evans blue passage to the brain were blocked by timolol (beta-adrenoceptor antagonist) but not by metoprolol (selective beta1-adrenoceptor antagonist). When given after the application of cold, clenbuterol was also able to reduce Evans blue and water content in the brain. Isoprenaline (beta-adrenoceptor agonist that does not cross the blood-brain barrier) showed a reduction in Evans blue extravasation only when given intracerebroventricularly. Vinblastine (a drug that prevents vesicle formation) produced a reduction of the amount of pinocytotic vesicles. We conclude that there is an influence of the central adrenergic nervous system on the formation and/or resolution of vasogenic brain oedema and that the alterations on water movement and Evans blue transport mediated by adrenergic drugs seem to be due, at least in part, to alterations of pinocytotic activity in capillary endothelial cells.     

Adrenergic influences on the control of blood-brain barrier permeabilit

Summary The central adrenergic innervation of the cerebral microvessels may play a role in the control of blood-brain barrier permeability. To pursue the study of this hypothesis we investigated the effect of noradrenaline on both the permeability of the blood-brain barrier to sodium fluorescein and on the pinocytotic activity of cerebral endothelial cells in the rat. Noradrenaline, stereotactically injected in the right lateral cerebral ventricle, significantly increased the cerebral extraction ratio of sodium fluorescein in a dose-dependent way. The same effect was induced by phenylephrine. Prostaglandin F₂ had no significant effect on the passage of sodium fluorescein through the blood-brain barrier.The effect of noradrenaline (150 µg) on the cerebral extraction ratio of sodium fluorescein was totally blocked by phenoxybenzamine (25 mg/kg i.p., 24 h before noradrenaline). Noradrenaline (150 µg) significantly increased the pinocytotic activity of cerebral endothelial cells. Phenoxybenzamine (as above) reduced the effect of noradrenaline on pinocytosis.It is concluded that noradrenaline increases the blood-brain barrier's permeability to sodium fluorescein, most probably through an effect on alpha adrenoceptors. The increase induced in the blood-brain barrier's permeability by noradrenaline seems to be due, at least in part, to an increase in the pinocytotic activity of endothelial cells. Send offprint requests to A. Sarmento at the above address