A new principle for tight junction modulation based on occludin peptides

The aim of this study was to investigate whether peptides from the extracellular loops of the tight junction protein occludin could be used as a new principle for tight junction modulation. Peptides of 4 to 47 amino acids in length and covering the two extracellular loops of the tight junction protein occludin were synthesized, and their effect on the tight junction permeability in Caco-2 cells was investigated using [C-14] mannitol as a paracellular marker. Lipopeptide derivatives of one of the active occludin peptides (OPs), synthesized by adding a lipoamino acid containing 14 carbon atoms (C-14-) to the N terminus of the peptide, were also investigated. Peptides corresponding to the N terminus of the first extracellular loop of occludin increased the permeability of the tight junctions without causing short-term toxicity. However, the peptides had an effect only when added to the basolateral side of the cells, which could be partly explained by degradation by apical peptidases and aggregate formation. By contrast, the lipopeptide C-14-OP90-103, which protects the peptide from degradation and aggregation, displayed a rapid apical effect. The L- and D-diastereomers of C-14-OP90-103 had distinctly different effects. The D-isomer, which releases intact OP90-103 from the lipoamino acid, displayed a rapid and transient increase in tight junction permeability. The L- isomer, which releases OP90-103 more rapidly, gave a more sustained increase in tight junction permeability. In conclusion, C-14-OP90-103 represents a prototype of a new class of tight junction modulators that act on the extracellular domains of tight junction proteins.

Treatment of maternal syphilis in rural South Africa: effect of multiple doses of benzathine penicillin on pregnancy loss

OBJECTIVES Despite few data, the treatment of syphilis in pregnant women using a single dose of benzathine penicillin is the standard of care in many resource-poor settings. We examined the effect of various doses of benzathine penicillin on pregnancy loss among women with a positive Rapid Plasma Reagin (RPR) test result in a rural South African district. METHODS All pregnant women making their first antenatal care visit during pregnancy were screened for syphilis using the RPR test. Those testing positive were counselled to receive three weekly doses of benzathine penicillin, and received a partner notification card. Pregnancy outcomes were determined from facility records or home visits where necessary. RESULTS Of 8917 women screened, 1043 (12%) had reactive syphilis serology; of those with titre data available, 30% had titres of 1:8 or greater. While 41% (n = 430) of women received all three doses as counselled, 30% (n = 312) received only one dose, and 20% (n = 207) did not return to the clinic to receive treatment. Among the 947 women with pregnancy outcome data available, there were 17 miscarriages and 48 perinatal deaths observed. There was a strong trend towards reduced risk of pregnancy loss among women receiving multiple doses of penicillin (adjusted OR for perinatal mortality for each additional dose received, 0.63; 95% CI, 0.48-0.84). CONCLUSIONS While this association requires further investigation, these results suggest that there may be substantial benefit to providing multiple doses of benzathine penicillin to treat maternal syphilis in this setting.

Incorporation of ovalbumin into ISCOMs and related colloidal particles prepared by the lipid film hydration method

The ann of this study was to investigate the incorporation of a model antigen, fluorescently labelled ovalbumin (FITC-OVA), into various colloidal particles including immune stimulating complexes (ISCOMs), liposomes, ring and worm-like micelles, lamellae and lipidic/layered structures that are formed from various combinations of the triterpene saponin Quil A, cholesterol and phosphatidylethanolamine (PE) following hydration of PE/cholesterol lipid films with aqueous Solutions of Quil A. Colloidal dispersions of these three components were also prepared by the dialysis method for comparison. FITC-OVA was conjugated with palmitic acid (P) and PE to produce P-FITC-OVA and PE-FITC-OVA, respectively. Both P-FITC-OVA and PE-FITC-OVA could be incorporated in all colloidal structures whereas FITC-OVA was incorporated only into liposomes. The incorporation of PE-FITC-OVA into all colloidal structures was significantly higher than P-FITC-OVA (P < 0.05). The degree of incorporation of protein was in the order: ring and worm-like micelles < liposomes and lipidic/layered structures < ISCOMs and lamellae. The incorporation of protein into the various particles prepared by the lipid film hydration method was similar to those for colloidal particles prepared by the dialysis method (provided both methods lead to the formation of the same colloidal structures). In the case of different colloidal structures arising due to the preparation method, differences in encapsulation efficiency were found (P < 0.05) for formulations with the same polar lipid composition. This study demonstrates that the various colloidal particles formed as a result of hydrating PE/cholesterol lipid films with different amounts of Quil A are capable of incorporating antigen, provided it is amphipathic. Some of these colloidal particles may be used as effective vaccine delivery systems. (C) 2004 Elsevier B.V. All rights reserved.

Pseudo-ternary phase diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by the lipid-film hydration method

Pseudo-ternary phase diagrams of the polar lipids Quil A, cholesterol (Chol) and phosphatidylcholine (PC) in aqueous mixtures prepared by the lipid film hydration method (where dried lipid film of phospholipids and cholesterol are hydrated by an aqueous solution of Quil A) were investigated in terms of the types of particulate structures formed therein. Negative staining transmission electron microscopy and polarized light microscopy were used to characterize the colloidal and coarse dispersed particles present in the systems. Pseudo-ternary phase diagrams were established for lipid mixtures hydrated in water and in Tris buffer (pH 7.4). The effect of equilibration time was also studied with respect to systems hydrated in water where the samples were stored for 2 months at 4degreesC. Depending on the mass ratio of Quil A, Chol and PC in the systems, various colloidal particles including ISCOM matrices, liposomes, ring-like micelles and worm-like micelles were observed. Other colloidal particles were also observed as minor structures in the presence of these predominant colloids including helices, layered structures and lamellae (hexagonal pattern of ring-like micelles). In terms of the conditions which appeared to promote the formation of ISCOM matrices, the area of the phase diagrams associated with systems containing these structures increased in the order: hydrated in water/short equilibration period < hydrated in buffer/short equilibration period < hydrated in water/prolonged equilibration period. ISCOM matrices appeared to form over time from samples, which initially contained a high concentration of ring-like micelles suggesting that these colloidal structures may be precursors to ISCOM matrix formation. Helices were also frequently found in samples containing ISCOM matrices as a minor colloidal structure. Equilibration time and presence of buffer salts also promoted the formation of liposomes in systems not containing Quil A. These parameters however, did not appear to significantly affect the occurrence and predominance of other structures present in the pseudo-binary systems containing Quil A. Pseudo-ternary phase diagrams of PC, Chol and Quil A are important to identify combinations which will produce different colloidal structures, particularly ISCOM matrices, by the method of lipid film hydration. Colloidal structures comprising these three components are readily prepared by hydration of dried lipid films and may have application in vaccine delivery where the functionality of ISCOMs has clearly been demonstrated. (C) 2003 Elsevier B.V. All rights reserved.

A comparison of pseudo-ternary diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by lipid-film hydration and dialysis

Pseudo-ternary diagrams for Quil A, phospholipid (phosphatidylcholine (PC) or phosphatidylethanolamine (PE)) and cholesterol were established in order to identify combinations that result in the formation of immune-stimulating complex (ISCOM) matrices and other colloidal structures produced by these three components in aqueous systems following lipid-film hydration or dialysis (methods that can be used to produce ISCOMs). In addition, the effect of equilibration time (1 month at 4degreesC) on the structures formed by the various combinations of the three components was investigated. Depending on the ratio of Quil A, cholesterol and phospholipid, different colloidal particles, including ISCOM matrices, liposomes and ring-like micelles, were found irrespective of the preparation method used. In contrast, worm-like micelles were only observed in systems prepared by lipid-film hydration. For samples prepared by dialysis, ISCOM matrices were predominantly found near the Quil A apex of the pseudo-ternary diagram (> 50% Quil A). On the other hand, for samples prepared by lipid-film hydration, ISCOM matrices were predominantly found near the phospholipid apex of the pseudo-ternary diagram (> 50% phospholipid). The regions in the pseudo-ternary diagrams in which ISCOM matrices were observed increased following an extended equilibration time, particularly for samples prepared by lipid-film hydration. Differences were also observed between pseudoternary diagrams prepared using either PE or PC as phospholipids.

Uptake of antigen encapsulated in polyethylcyanoacrylate nanoparticles by D1-dendritic cells

Polyethylcyanoacrylate (PECA) nanoparticles were prepared by interfacial polymerization of a water-in-oil microemulsion. Nanoparticles were isolated from the polymerization template by sequential ethanol washing and centrifugation. A nanocapsule preparation yielding the original particle size and distribution following redispersion in an aqueous solution was achieved by freeze-drying the isolated nanoparticles in a solution of 5% w/v sugar. The cytotoxicity and uptake of nanocapsules by dendritic cells was investigated using a murine-derived cell line (D1). PECA nanoparticles were found to adversely effect cell viability at concentrations greater than 10 mug/ml of polymer in the culture medium. In comparison to antigen in solution, cell uptake of antigen encapsulated within nanoparticles was significantly higher at both 4 and 37 degreesC. Following a 24 h incubation period, the percentage of cells taking-up antigen was also increased when antigen was encapsulated in nanoparticles as compared to antigen in solution. The uptake of nanoparticles and the effect of antigen formulation on morphological cell changes indicative of cell maturation were also investigated by scanning electron microscopy (SEM). SEM clearly demonstrated the adherence of nanoparticles to the cell surface. Incubation of D1 dendritic cells with nanoparticles containing antigen also resulted in morphological changes indicative of cell maturation similar to that observed when the cells were incubated with lipopolysaccharide. In contrast, cells incubated with antigen solution did not demonstrate such morphological changes and appeared similar to immature cells that had not been exposed to antigen.

Assessing the effectiveness of a mammography screening service

Background: Trials have shown that mammography screening reduces mortality and probably decreases morbidity related to breast cancer. Methods: We assessed whether the major mammography service in Western Australia (BreastScreen WA) is likely to reduce mortality by comparing prognostic variables between screen-detected and other cases of breast cancer diagnosed in 1999. We assessed likely reductions in morbidity by comparing treatments received by these two groups. To confirm mortality and morbidity reduction, we also compared prognostic variables and treatments with targets. Information on demographic variables, tumour characteristics at presentation and treatments were collected from medical records for all incident cases of breast cancer in Western Australia in 1999. We matched cases with the Western Australian Cancer Registry records to determine which cases had been detected by BreastScreen WA. Results: BreastScreen WA achieved the targets for mortality reduction. Tumours detected by BreastScreen WA were smaller in size, less likely to have vascular invasion, of lower histological grade and were more likely to be ductal carcinoma in situ alone without invasive carcinoma. Oestrogen receptor status was more likely to be positive, the difference in progesterone status was not significant, and lymph node involvement tended to be lower. BreastScreen WA patients were treated more often with local therapy and less often with systemic therapy, and the proportion of patients treated with breast-conserving surgery was close to the target for minimizing morbidity in breast cancer. Conclusion: Mammographic detection of breast cancer by BreastScreen WA is associated with reduced breast cancer morbidity and a more favourable prognosis.

Steatosis: Co-factor in other liver diseases

The prevalence of fatty liver is rising in association with the global increase in obesity and type 2 diabetes. In the past, simple steatosis was regarded as benign, but the presence of another liver disease may provide a synergistic combination of steatosis, cellular adaptation, and oxidative damage that aggravates liver injury. In this review, a major focus is on the role of steatosis as a co-factor in chronic hepatitis C (HCV), where the mechanisms promoting fibrosis and the effect of weight reduction in minimizing liver injury have been most widely studied. Steatosis, obesity, and associated metabolic factors may also modulate the response to alcohol- and drug-induced liver disease and may be risk factors for the development of hepatocellular cancer. The pathogenesis of injury in obesity-related fatty liver disease involves a number of pathways, which are currently under investigation. Enhanced oxidative stress, increased susceptibility to apoptosis, and a dysregulated response to cellular injury have been implicated, and other components of the metabolic syndrome such as hyperinsulinernia and hyperglycemia are likely to have a role. Fibrosis also may be increased as a by-product of altered hepatocyte regeneration and activation of bipotential hepatic progenitor cells. In conclusion, active management of obesity and a reduction in steatosis may improve liver injury and decrease the progression of fibrosis.