A physico-chemical study on amphiphilic cyclodextrin/liposomes nanoassemblies with drug carrier potential.

In this paper, two medusa-like ACyDs, modified at the primary rim bearing four (ACyD4) and eight carbons (ACyD8) acyl chain length, and one bouquet-like CyD, modified at primary side with thiohexyl and at secondary one with oligoethylene moiety (SC6OH), were investigated for their ability to assemble in nanostructures or to form hybrid dipalmitoylphosphatidylcholine (DPPC)/ACyDs systems. The lipophilicity of these molecules and the different preparation methods used in this study (thin layer evaporation and nanoprecipitation method) significantly affect the aggregation behaviour in aqueous medium. Except for the shortest medusa-like ACyD4, the other ACyDs formed stable nanoaggregates for at least 45 days. The effect of ACyDs on the thermotropic behaviour of DPPC liposomes was also studied by differential scanning calorimetry analysis, thus elucidating their interaction with liposomes to afford hybrid liposome/ACyDs systems. The medusa-like ACyD4 cannot be used to realize nanosystems because it quickly aggregates or it induces a complete destabilization of the liposomes. At the highest concentration investigated (0.01 molar fraction), both ACyD8 and SC6OH interacted with DPPC liposomes, forming ACyD/DPPC or SC6OH/DPPC hybrid vesicular carriers.

The validity of nasal endoscopy in patients with chronic rhinosinusitis-An inter-rater agreement study.

OBJECTIVES: Nasal endoscopy is a cornerstone in diagnosing sinonasal disease, but different raters might generate different results using the technique. Our study aims to evaluate the agreement between multiple raters to assess the validity of nasal endoscopy. DESIGN/PARTICIPANTS: Three independent and blinded raters evaluated 28 patients (56 nasal cavities) diagnosed with chronic rhinosinusitis according to the European Position Paper on Rhinosinusitis and Nasal Polyps. The ratings were compared using unweighted Fleiss' kappa coefficients (Kf ) for each objective parameter. SETTING: The department of Otorhinolaryngology, Odense University Hospital, Denmark. MAIN OUTCOME MEASURES: The ratings were quantified in a modified Lund-Kennedy endoscopy score and focused on the objective parameters specified in the diagnostic criteria: polyps, oedema and discharge. RESULTS: The raters agreed on the findings concerning polyps and discharge but not regarding oedema with the inter-rater agreement for the different parameters being: polyps Kf =.66 (SE .07, P<.001), oedema Kf =.05 (SE .07, P=.21), discharge Kf =.35 (SE .08, P<.001), oedema exclusively in middle meatus Kf =-.07 (SE .04, P=.8) and discharge exclusively in middle meatus Kf =.16 (SE .07, P=.01). CONCLUSION: Using nasal endoscopy, the evaluation of polyps by multiple raters showed sufficient reliability indicating an acceptable objective evaluation. The evaluation of discharge achieved a fair level of agreement while the assessment of oedema could not achieve a sufficient reliability questioning the inclusion of oedema in the criteria for diagnosing sinonasal disease.

Primary and secondary restorative proctocolectomy for familial adenomatous polyposis: complications and long-term bowel function.

AIM: The aim of the study was to evaluate intra-operative difficulties, complications and long-term bowel function in polyposis patients undergoing conversion of an ileorectal anastomosis to an ileoanal pouch, compared with patients with a primary ileoanal pouch operation. METHOD: A national register-based retrospective study was performed with clinical follow-up and a questionnaire on long-term bowel function. RESULTS: There were 84 patients in the study: 59 (70%) had a primary pouch operation and in 25 (30%) a secondary pouch procedure was attempted. This was abandoned, in one case, leaving 24 patients who had a successful secondary restorative proctocolectomy. The median (range) follow-up was 123 (0-359) months. There were no intra-operative difficulties in the 59 primary operations, but intra-operative difficulties were reported in nine of 25 secondary operations (P < 0.001). Complications within 1 month of surgery occurred in six of 59 primary operations and in none of 24 secondary operations (P < 0.001); and late surgical complications occurred in eight of 55 primary operations and in eight of 24 secondary operations (P = 0.13). The only difference in bowel function was a lower frequency of nocturnal defaecation after secondary pouch formation (P = 0.02). CONCLUSION: Reoperation with proctectomy after a previous ileorectal anastomosis and conversion to restorative proctocolectomy is feasible in polyposis patients, with morbidity and functional results similar to those seen after a primary pouch operation.

Self-assembling microparticles with controllable disruption properties based on cyclodextrin interactions.

In this paper, we present the formation of particles by self-assembly of cyclodextrin polymers and hydrophobically modified dextran followed by a controlled disruption of the particles by addition of a trigger molecule competing for the cyclodextrin cavities. The produced particles are formed from poly(vinylpyrrolidone)-co-beta-cyclodextrin and dextran-benzoate, both biocompatible polymers, and are all in the nano-/micrometer range and hence suitable for drug delivery purposes. The particle formation was studied in different ratios of poly(vinylpyrrolidone)-co-beta-cyclodextrin and dextran-benzoate by visual inspections, dynamic light scattering, isothermal titration calorimetry and SEM. The triggering of particle disruption was achieved by addition of hydroxyadamantane which has a very strong affinity towards the beta-cyclodextrin cavities. The stepwise addition of hydroxyadamantane was followed by dynamic light scattering and SEM measurements, revealing a disruption of the particles due to the addition of this competitor. These particles are believed to be promising candidates for controlled drug delivery systems, due to their unique ability to disrupt in a controlled manner.

Structural background of cyclodextrin-protein interactions.

Cyclodextrins are cyclic oligosaccharides with the shape of a hollow truncated cone. Their exterior is hydrophilic and their cavity is hydrophobic, which gives cyclodextrins the ability to accommodate hydrophobic molecules/moieties in the cavity. This special molecular arrangement accounts for the variety of beneficial effects cyclodextrins have on proteins, which is widely used in pharmacological applications. We have studied the interaction between beta-cyclodextrin and four non-carbohydrate-binding model proteins: ubiquitin, chymotrypsin inhibitor 2 (CI2), S6 and insulin SerB9Asp by NMR spectroscopy at varying structural detail. We demonstrate that the interaction of beta-cyclodextrin and our model proteins takes place at specific sites on the protein surface, and that solvent accessibility of those sites is a necessary but not compelling condition for the occurrence of an interaction. If this behaviour can be generalized, it might explain the wide range of different effects of cyclodextrins on different proteins: aggregation suppression (if residues responsible for aggregation are highly solvent accessible), protection against degradation (if point of attack of a protease is sterically 'masked' by cyclodextrin), alteration of function (if residues involved in function are 'masked' by cyclodextrin). The exact effect of cyclodextrins on a given protein will always be related to the particular structure of this protein.

Two-step enzymatic synthesis of maltooligosaccharide esters.

Glucose and maltose esters were synthesised in organic media by employing a lipase (E.C. 3.1.1.3) from Candida antarctica. In a second reaction step, a transglycosylation catalysed by a cyclodextrin glycosyltransferase (E.C. 2.4.1.19) from either Paenibacillus sp. F8 or Bacillus sp. strain no. 169 (DSM 2518) extended the degree of polymerisation (DP) of the carbohydrate moieties of the carbohydrate esters. The donor substrates used were either a cyclodextrin, a maltooligosaccharide or starch. The highest rate of low DP maltooligosaccharide ester formation was obtained when starch was used as glycosyl donor and caproyl maltose as glycosyl acceptor. The structures of two of the products were identified by 1H and 13C NMR and MALDI-TOF MS as capronate monoesters of maltotriose and maltotetraose, with the ester bond at C-6 of the second glucose unit from the reducing end.

Suitability and limitations of methods for characterisation of activity of malto-oligosaccharide-forming amylases.

The suitability and limitations of essential methods and reference substrates used for characterisation of activity of amylolytic enzymes is investigated. Saccharogenic, chromogenic and chromatographic methods are included. The results are discussed in relation to the measurement of reaction rates, determination of action mode and product specificity and the impact on identification and nomenclature of malto-oligosaccharide-forming amylases. An accurate determination of reaction rates using the saccharogenic methods strongly depends on the degree of polymerisation (DP) of the standards used and the hydrolysis products formed by the amylase. Particularly the use of glucose as standard can lead to overestimates due to the differences in the reducing potential of glucose and malto-oligosaccharides. The reliability of the chromogenic methods for determination of action mode depends on the DP of the substrate and the specificity of the amylase. For a characterisation of the starch hydrolysis products and the variation in the DP during hydrolysis, high performance anion-exchange chromatography with pulsed amperometric detection provided a fast and reliable method. A literature survey revealed varying and inconsistent use of nomenclature of malto-oligosaccharide forming amylases. Therefore a systematic approach identifying three main classes of activity is suggested using not only the mode of action and the DP of the major product but also the stage of hydrolysis at which this product is formed.

Rapid detection of malto-oligosaccharide-forming bacterial amylases by high performance anion-exchange chromatography.

High performance anion-exchange chromatography with pulsed amperometric detection was applied for the rapid analysis of malto-oligosaccharides formed by extracellular enzyme preparations from 49 starch-degrading bacterial strains isolated from soil and compost samples. Malto-oligosaccharide-forming amylases, indicated by a predominant formation of maltohexaose from starch, were produced by enzyme preparations from four of the isolates growing at pH 7.0 and 10.

Azathioprine and 6-mercaptopurine for the treatment of perianal Crohn's disease in children.

Numerous adult studies show a 30-65% response rate to azathioprine (AZA) or 6-mercaptopurine (6-MP) for significant perianal Crohn's disease. The aim of this study was to evaluate whether these drugs healed pediatric perianal Crohn's disease. Records of pediatric Crohn's patients were retrospectively reviewed for significant perianal disease treated with AZA or 6-MP for > or =6 months. The patient's perianal disease was reviewed and evaluated for fistulas, drainage, induration, and tenderness. In addition, the patients were given a score using the Irvine Perianal Disease Activity Index (PDAI). Patients were retrospectively scored upon initiation of treatment and after six months of therapy. Possible scores ranged from 0-20. Twenty patients met the study criteria. Five patients were considered treatment failures. One patient required a colostomy after 1.5 months of therapy, one developed pancreatitis, and three were noncompliant with therapy. Of the remaining 15 patients who were treated for > or =6 months, 67% had an improvement in drainage, 73% in tenderness, 60% in induration, and 40% in fistula closure. The mean Irvine PDAI was 7.67 +/- 2.19 initially and 4.40 +/- 1.72 after six months of therapy. The improvement was statistically significant (p < 0.001). AZA and 6-MP are effective treatments for healing significant perianal Crohn's disease in pediatrics.

Production of cyclomaltononaose (delta-cyclodextrin) by cyclodextrin glycosyltransferases from Bacillus spp. and bacterial isolates.

The conversion of soluble starch to cyclomaltohexaose (alpha-CD), cyclomaltoheptaose (beta-CD), cyclomaltooctaose (gamma-CD) and cyclomaltononaose (delta-CD) by cyclodextrin glycosyltransferases (E.C. 2.4.1.19) from Bacillus spp. and bacterial isolates was studied. The results show that delta-CD was formed by all the enzymes investigated in the range of 5%-11.5% of the total amount of alpha-, beta-, gamma-, and delta-CD produced.

Immuno-capillary electrophoresis for the characterization of a monoclonal antibody against DNA.

A monoclonal antibody against DNA established from a mouse strain that spontaneously develops systemic lupus erythematosus was characterized by migration shift immuno-capillary electrophoresis. The minimal size for DNA binding antibody was > 16 bases and the interaction with a double-stranded 32-mer oligonucleotide was almost one order of magnitude stronger than the interaction with a single-stranded oligonucleotide. The binding was highly dependent on the ionic strength conditions with an increase in binding with a decrease in ionic strength. The estimate of the dissociation constant for the antibody binding of a single stranded 32-mer oligonucleotide was 0.62 microM at pH 7.90. This value was in good agreement with the value of 0.44 microM measured by an independent method using biosensor (surface plasmon resonance) technology.

Effects of illumination intensity on bacteriochlorophyllc homolog distribution inChloroflexus aurantiacus grown under controlled conditions.

Green photosynthetic bacteria contain a mixture of stereoisomers and homologs of their major light harvesting pigment, bacteriochlorophyll (BChl)c. We have determined the distribution of photosynthetic pigments in the green filamentous bacteriumChloroflexus aurantiacus grown in turbidostat culture under light-limited conditions at 5 different illumination intensities. Pigments were extracted from isolated cells, analyzed by HPLC, and the homologs of BChlc identified by their mass spectra. The ratio between BChlc, BChla and carotenoid remained constant at low illumination intensities; at higher intensities BChla and carotenoid increased in parallel compared to BChlc. The BChlc homolog distribution changed even under conditions where the ratio of the total amount to the other pigments was unchanged, but there were no evidence for a constant stoichiometric ratio between any pair of homologs.

Responses and molecular heterogeneity of IR-GIP after intraduodenal glucose and fat.

The plasma responses and molecular heterogeneity of human immunoreactive gastric inhibitory polypeptide (IR-GIP) after intraduodenal administration of equicaloric amounts of glucose (25 g) and fat (10 g) were determined with five different antisera. Two of these do not cross-react with 8 kdaltons (kDa) IR-GIP. In the fasting state plasma IR-GIP concentrations were highest measured with antisera, which detected both 5 and 8 kDa IR-GIP. The total integrated or incremental IR-GIP areas were similar after glucose and fat when determined with the same antiserum. When antisera with similar cross-reactivity with human 5 kDa IR-GIP were compared, no difference in incremental IR-GIP areas after either stimulus was found between antisera that did and did not measure 8 kDa IR-GIP. Gel filtration of plasma showed a consistent increase in 5 kDa IR-GIP 60 min after both glucose and fat but small and inconsistent changes in 8 kDa IR-GIP. Thus fat on a weight and molar basis is more potent than glucose in releasing IR-GIP. Both fat and glucose release predominantly the 5 kDa IR-GIP. Although of importance for the absolute IR-GIP level in plasma, 8 kDa IR-GIP contributes little to the increase in IR-GIP after both stimuli.