Prebiotics as excipients for enhancement of stability and functionality of Bifidobacterium longum ssp. infantis with potential application as symbiotics in food and pharmaceuticals.

Objective: Formulations containing probiotics are promoted due to health benefits. During lyophilization and subsequent storage in the gastrointestinal tract, bacteria are exposed to stress conditions that can lead to impairment and loss of viability. Methods: The suitability of various excipients for enhancing the stability and functionality of Bifidobacterium longum subsp. infantis during storage as freeze-dried powder and through exposure to acid and bile was investigated. Cells were lyophilized in the presence of sucrose, trehalose, lactose, cellobiose and fructooligosaccharide (FOS) and stored at 4 °C or 25 °C. The effect of diverse protectants on the persistence after exposure to acid and bile environment was examined through determination of the colony forming units, the ß-glucosidase and ß-galactosidase activity and the membrane integrity changes. Results: Cells freeze-dried in the presence of cryoprotectants had comparable survivability during storage at 4 °C whereas the survival rate at 25 °C of cells protected by cellobiose and FOS was higher than for those protected with sucrose and trehalose. Furthermore, the respective excipients used as cryoprotectants enhanced the stability of cells exposed to simulated gastric and small intestinal medium. Stabilization may be achieved through different mechanism of action such as protecting the membrane integrity and as metabolizable substrates. Overall, prebiotic and thus metabolizable protectants including cellobiose and FOS were superior to other protectants used. Conclusion: In symbiotic formulas with B. infantis, these sugars might serve as prebiotics and stabilizers of this probiotic strain during lyophilization, storage and in gastrointestinal conditions simultaneously, potentially increasing its health-promoting effects.

[Direct and indirect costs of fractures due to osteoporosis in Austria]. / Direkte und indirekte Kosten von osteoporotisch bedingten Frakturen in Österreich.

OBJECTIVES: We examined the financial burden of osteoporosis in Austria. METHODS: We took both direct and indirect costs into consideration. Direct costs encompass medical costs such as expenses for pharmaceuticals, inpatient and outpatient medical care costs, as well as other medical services (e.g., occupational therapies). Non-medical direct costs include transportation costs and medical devices (e.g., wheel chairs or crutches). Indirect costs refer to costs of productivity losses due to absence of work. Moreover, we included costs for early retirement and opportunity costs of informal care provided by family members. While there exist similar studies for other countries, this is the first comprehensive study for Austria. For our analysis, we combined data of official statistics, expert estimates as well as unique patient surveys that are currently conducted in the course of an international osteoporotic fracture study in Austria. RESULTS: Our estimation of the total annual costs in the year 2008 imposed by osteoporosis in Austria is 707.4 million €. The largest fraction of this amount is incurred by acute hospital treatment. Another significant figure, accounting for 29% of total costs, is the opportunity cost of informal care. CONCLUSIONS: The financial burden of osteoporosis in Austria is substantial. Economic evaluations of preventive and therapeutic interventions for the specific context of Austria are needed to inform health policy decision makers.

Phenotype-related differential alpha-2,6- or alpha-2,3-sialylation of glycoprotein N-glycans in human chondrocytes.

OBJECTIVE: Sialic acids frequently occur at the terminal positions of glycoprotein N-glycans present at chondrocyte surfaces or in the cartilage matrix. Sialic acids are transferred to glycoproteins in either alpha-2,3 or alpha-2,6 linkage by specific sialyltransferases (SiaTs) and can potentially affect cell functions and cell-matrix interactions. The present study aimed to assess the relationship between the expression of the human chondrocyte phenotype and the sialylation of chondrocyte glycoprotein N-glycans. METHODS: The transcription of 5 SiaT was quantified using real-time Reverse transcription polymerase chain reaction (RT-PCR) assays. N-glycan analysis was performed using LC-ESI-MS. Primary human chondrocytes were cultured in monolayer or alginate beads and compared to the chondrocyte cell lines C-28/I2 and SW1353. In addition, effects of interleukin-1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) on primary cells were assessed. RESULTS: Primary human chondrocytes predominantly express alpha-2,6-specific SiaTs and accordingly, alpha-2,6-linked sialic acid residues in glycoprotein N-glycans. In contrast, the preponderance of alpha-2,3-linked sialyl residues and, correspondingly, reduced levels of alpha-2,6-specific SiaTs are associated with the altered chondrocyte phenotype of C-28/I2 and SW1353 cells. Importantly, a considerable shift towards alpha-2,3-linked sialic acids and alpha-2,3-specific SiaT mRNA levels occurred in primary chondrocytes treated with IL-1beta or tumour necrosis factor-alpha (TNF-alpha). CONCLUSION: The expression of the differentiated chondrocyte phenotype is linked to the ratio of alpha-2,6- to alpha-2,3-linked sialic acids in chondrocyte glycoprotein N-glycans. A shift towards altered sialylation might contribute to impaired cell-matrix interactions in disease conditions.

Comparison between chondroprotective effects of glucosamine, curcumin, and diacerein in IL-1beta-stimulated C-28/I2 chondrocytes.

OBJECTIVE: To compare the effects of glucosamine (GlcN), curcumin, and diacerein in immortalized human C-28/I2 chondrocytes at the cellular and the gene expression level. This study aimed to provide insights into the proposed beneficial effects of these agents and to assess the applicability of the C-28/I2 cell line as a model for the evaluation of chondroprotective action. METHODS: Interleukin-1beta (IL-1beta)-stimulated C-28/I2 cells were cultured in the presence of GlcN, curcumin, and diacerein prior to the evaluation of parameters such as viability, morphology and proliferation. The impact of GlcN, curcumin, and diacerein on gene expression was determined using quantitative real-time RT-PCR (qPCR). RESULTS: At the transcriptional level, 5 mM GlcN and 50 microM diacerein increased the expression of cartilage-specific genes such as aggrecan (AGC) and collagen type II (COL2), while reducing collagen type I (COL1) mRNA levels. Moreover, the IL-1beta-mediated shift in gene expression pattern was antagonized by GlcN and diacerein. These effects were associated with a significant reduction in cellular proliferation and the development of chondrocyte-specific cell morphology. In contrast, curcumin was not effective at lower concentrations but even damaged the cells at higher amounts. CONCLUSIONS: Both GlcN and diacerein promoted a differentiated chondrocytic phenotype of immortalized human C-28/I2 chondrocytes by altering proliferation, morphology, and COL2/COL1 mRNA ratios. Moreover, both agents antagonized inhibitory effects of IL-1beta by enhancing AGC and COL2 as well as by reducing COL1 mRNA levels.

Preclinical In Vitro and In Vivo Evaluation of [18F]FE@SUPPY for Cancer PET Imaging: Limitations of a Xenograft Model for Colorectal Cancer.

Molecular imaging probes such as PET-tracers have the potential to improve the accuracy of tumor characterization by directly visualizing the biochemical situation. Thus, molecular changes can be detected early before morphological manifestation. The A3 adenosine receptor (A3AR) is described to be highly expressed in colon cancer cell lines and human colorectal cancer (CRC), suggesting this receptor as a tumor marker. The aim of this preclinical study was the evaluation of [18F]FE@SUPPY as a PET-tracer for CRC using in vitro imaging and in vivo PET imaging. First, affinity and selectivity of FE@SUPPY and its metabolites were determined, proving the favorable binding profile of FE@SUPPY. The human adenocarcinoma cell line HT-29 was characterized regarding its hA3AR expression and was subsequently chosen as tumor graft. Promising results regarding the potential of [18F]FE@SUPPY as a PET-tracer for CRC imaging were obtained by autoradiography as ≥2.3-fold higher accumulation of [18F]FE@SUPPY was found in CRC tissue compared to adjacent healthy colon tissue from the same patient. Nevertheless, first in vivo studies using HT-29 xenografts showed insufficient tumor uptake due to (1) poor conservation of target expression in xenografts and (2) unfavorable pharmacokinetics of [18F]FE@SUPPY in mice. We therefore conclude that HT-29 xenografts are not adequate to visualize hA3ARs using [18F]FE@SUPPY.

Lectin binding studies on C-28/I2 and T/C-28a2 chondrocytes provide a basis for new tissue engineering and drug delivery perspectives in cartilage research.

The present study was performed to evaluate the applicability of plant lectins as mediators of bioadhesion in cartilage research using human chondrocyte cell lines C-28/I2 and T/C-28a2. The bioadhesive properties of fluorescein-labelled lectins with different carbohydrate specificities were investigated by flow cytometry. Specificity of the lectin-cell interactions was ascertained by competitive inhibition using complementary carbohydrates. As compared to that of other lectins, the interaction between wheat germ agglutinin (WGA) and chondrocytic cells was characterised by remarkable cytoadhesion, adequate binding strength and a high degree of specificity for N-acetyl-glucosamine as contained in hyaluronan chains. We therefore suggest WGA to be a promising candidate for mediating bioadhesion to low-adhesive scaffolds in cartilage tissue engineering. Moreover, the WGA-association rate of C-28/I2 and T/C-28a2 cells was dependent on temperature indicating cellular uptake of membrane-bound WGA. Intracellular enrichment was confirmed by confocal microscopy. Equilibration of intracellular pH gradients with monensin resulted in the reversal of quenching effects indicating accumulation of WGA within acid compartments of chondrocytic cells. Thus, WGA might be internalised into chondrocytes together with hyaluronan via the CD44 receptor-mediated endocytosis pathway and accumulated within lysosomes. This physiological process could represent a feasible pathway to target WGA-functionalised drug delivery devices into chondrocytes.

Vaginal Lactobacillus microbiota of healthy women in the late first trimester of pregnancy.

OBJECTIVE: This study was undertaken to characterise the dominant species of Lactobacillus colonising the vagina of healthy pregnant women, to examine some of their phenotypic and genotypic properties, and to gain a better understanding of the potential role of species, which might be associated with infection-free status. DESIGN: A prospective descriptive cohort study. SETTING: Department of Obstetrics and Gynaecology, Medical University of Vienna and Medical School, Vienna, Austria. SAMPLE: A total of 200 women in the late first trimester of pregnancy without clinical signs of vaginal infection were included in the study. Of these, 126 women were found to have a normal vaginal flora based on Gram stain. METHODS: Culture probes from those 126 women were further processed for identification of Lactobacillus species. Overall, 168 colonies from 84 women were identified as belonging to the Lactobacillus genus. Based on the combined results of microbiological methods and genus-specific, multiplex, and species-specific polymerase chain reaction, lactobacilli were recovered from 72 women. MAIN OUTCOME MEASURES: Identification of Lactobacillus species of the vaginal flora of healthy pregnant women. RESULTS: The most frequently occurring species were Lactobacillus crispatus and Lactobacillus gasseri, followed by Lactobacillus jensenii and Lactobacillus rhamnosus. CONCLUSIONS: Our results may have implications on the composition and on the use of Lactobacillus preparations for the prevention of recurrent vaginal infection.

Deficiences in phenotype expression and function of dentritic cells from patients with early breast cancer.

PURPOSE: Monocytes derived from patients with early breast cancer (EBC) have shown functional deficiencies. These functional deficiencies are characterized by changes in phenotype and morphology. We have expanded these investigations to dendritic cells generated from monocytes from patients with early breast cancer. - PATIENTS AND METHODS: Peripheral blood from 36 patients with EBC and from 26 healthy age-matched women was drawn and prepared for ex vivo generation of dendritic cells (DC) by incubation with granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin 4 (IL4). The phenotype of DC was examined by flow-cytometry. T cell - proliferation was induced with tetanus toxoid pulsed autologous dendritic cell. - RESULTS: Dendritic cells generated from monocytes from EBC-patients showed a significantly lower expression of the phenotype-associated antigens CD1a, CD83, CD80, CD86 and CD54 than the dendritic cells from healthy controls. T cell - proliferation in response to TT-pulsed autologous dendritic cells was significantly decreased when induced with dendritic cells from patients with early breast cancer, when compared to healthy controls. Morphologically, only dendritic cells from healthy women possessed prominent dendrites indicating maturity. - CONCLUSIONS: These findings indicate that dendritic cells generated from monocytes from patients with early breast cancer express an immature phenotype, exhibit immature morphology and show functional deficits when compared to the cells derived from healthy age-matched controls. Whether these findings offer a potential target for therapeutic interventions remains to be elucidated.

Evaluation of mitoxantrone-loaded albumin microspheres following intraperitoneal administration to rats.

Mitoxantrone has demonstrated therapeutic efficacy in the regional treatment of intraperitoneal malignancies. However, severe local toxicity was dose limiting. Consequently, a new injectable sustained delivery formulation of mitoxantrone has been developed: the drug was incorporated (8.2 w/w%) in highly hydrophilic albumin microspheres. In vitro drug release profile was modified by matrix crosslinking extent. The extractable amount of residual crosslinking agent (glutaraldehyde) in the microspheres was lower than 6 ppm. Mitoxantrone concentration in peritoneal fluid and plasma was determined up to 72 h after intraperitoneal administration of 30, 60 and 120 mg mitoxantrone per m2 body surface area as solution and in the form of a dispersion containing mitoxantrone-loaded microspheres to rats. Data analysis revealed sustained release of mitoxantrone from microspheres into peritoneal fluid in all dosage groups. The initial high drug levels in peritoneal fluid and plasma observed after application of mitoxantrone in the solution form were prevented by administration of the drug incorporated in microspheres. However, tumoricidal drug levels in peritoneal fluid were maintained over a comparable time span. In addition, preliminary toxicity data suggest a superior local tolerability of mitoxantrone-loaded microspheres. The dose of intraperitoneally administered mitoxantrone might be increased from 30 to 60 mg per m2 body surface area using the slow release formulation. In conclusion, the described microsphere drug delivery system for mitoxantrone might overcome dose-limiting drug toxicity.

Pharmacological actions of central nervous system depressants given intravenously as suspensions.

Three central nervous system (CNS) depressants, methohexital (sodium), midazolam (maleate), and flunitrazepam, were given intravenously to rabbits in equimolar doses as suspensions and solutions. The mean diameters of the particles in the suspensions were 770, 840, and 460 nm, respectively. The CNS actions were verified by recording the bioelectrical activity of the precentral cortex. The suspensions were tolerated well. There were no differences in onset, duration, and maximal intensity of action between suspension and solution of each drug. The results of this study should facilitate pharmacological studies of drugs acting on the CNS that are insoluble in water.

Optimization of a formulation containing viable lactic acid bacteria.

In the present study, gastric juice resistant tablet formulations of lactic acid bacteria (LAB) were developed, using hydroxypropylmethylcellulose acetate succinate (HPMCAS) as well as alginates, apple pectin and Metolose as matrix forming components. To optimize the formulation-using survival rate in acid medium, and disintegration time in intestinal fluid as test parameters-tablets were modified with respect to LAB content, amount of applied excipients per tablet, and compaction forces. A decrease of viable cells of not more than one log unit after 2h of incubation in acid medium was desired, as well as a disintegration time of 1h in phosphate buffer pH 6.8. It was found that the amount of HPMCAS in the tablet correlates with gastric juice resistance. As HPMCAS also leads to a decrease of disintegration time in intestinal fluid, slight amounts of this excipient were preferred. The best protective qualities against artificial gastric juice were observed when tablets were prepared from compaction mixtures of LAB, HPMCAS and sodium alginate.

Action of pyrethrum-based formulations against grain weevils.

Pyrethrum extract, containing six insecticidal esters, has a long history of successful application in the control of stored products. Its low environmental hazard makes it an ideal pesticide for outdoor pre-harvest treatment. However the disadvantage of its low light stability then becomes apparent. This drawback can be overcome by the complexation of pyrethrum extract with gamma-cyclodextrin. Primary object of the conducted studies was to investigate the effect of complexation upon the insecticidal action against the grain weevil, an important storage pest in temperate climates. To slow down the quick metabolism of pyrethrum by the insects' microsomal system synergistic substances are added. Additional to the already well-known piperonyl butoxide two natural synergists, sesamol and tocopherol acetate, were combined with pyrethrum extract to investigate their synergistic activity. A complex of pyrethrum with gamma-cyclodextrin, with piperonyl butoxide as synergist, has a slightly enhanced action compared to a commercial product, which contained pyrethrum in its free form. Sesamol and tocopherol acetate also display a synergistic action, but to a much smaller degree, even if applied in larger amounts. The optimal concentration of pyrethrum was found to be 0.3% combined with 3% piperonyl butoxide.

Toxicity of a particulate formulation for the intraperitoneal application of mitoxantrone.

Mitoxantrone (MXN) has demonstrated therapeutic efficacy in the intraperitoneal treatment of malignancies. However, severe local toxicity is dose limiting. Therefore, a particulate formulation of MXN, the drug incorporated in albumin microspheres, was evaluated concerning tolerability. Survival rates as well as alterations in body weight, food intake, water intake, urine volume, urine specific gravity, urine protein content, and complete blood count were observed following single or multiple intraperitoneal injections of MXN solution, dispersions containing MXN-loaded microspheres or unloaded microspheres, and the injection vehicle to female and male Sprague-Dawley rats. Applied MXN dosage was equivalent to 30 mg/m2 body surface area. Unloaded microspheres were well tolerated without signs of toxicity. Application of MXN solution or MXN-loaded microspheres resulted in similar survival rates (56% 9 weeks after single injection) and in a comparable bone marrow toxicity (mainly leucopenia). Body weight, food and water intake as well as urine volume were decreased following application of MXN solution, whereas a progressive gain in weight and no remarkable alterations in nutrition and urine excretion were noted after administration of MXN-loaded and unloaded microspheres, or of the injection vehicle. In conclusion, intraperitoneal injection of MXN incorporated in albumin microspheres exhibits in part less toxicity than conventional treatment.

Similar central actions of intravenous methohexitone suspension and solution in the rabbit.

The central actions of solutions of methohexitone sodium and suspensions of methohexitone given intravenously in equimolar doses have been compared. The administrations induced identical anaesthesia. Because, in general, drugs acting upon the central nervous system are poorly soluble in water, this finding should be useful for routine pharmacological investigations as it avoids the use of organic solvents which may themselves affect the central nervous system.

Strategies for the evaluation and selection of potential vaginal probiotics from human sources: an exemplary study.

During the last years, the application of probiotics in gynaecological clinical practice has gained increasing relevance regarding therapy and prevention. This trend has also provoked the need for having tailored pharmaceutical preparations containing powerful microbial strains with defined properties. For the development of such preparations, several factors and criteria have to be considered, thereby not only focusing on identity and safety aspects as well as individual properties of the bacterial strains, but also on technological issues, such as stability and targeted release from the preparation. Against this background, this report exemplarily addresses the development procedure of a probiotic bacterial formulation for gynaecological application, covering the search for suitable strains, assessing their microbiological, molecular biological and physiological characterisation, and the selection for their use in clinical trials. In detail, starting with 127 presumptive lactobacilli isolates of vaginal origin, a step-by-step selection of candidate strains meeting special criteria was thoroughly examined, finally leading to a preparation consisting of four individual Lactobacillus strains that possess particular significance in women's urogenital health. Relevant issues and quality criteria of probiotic preparations used in gynecology are addressed and exemplarily introduced.

Radiosynthesis of [11C]SNAP-7941--the first PET-tracer for the melanin concentrating hormone receptor 1 (MCHR1).

The melanin concentrating hormone (MCH) system is a new target to treat human disorders. Our aim was the preparation of the first PET-tracer for the MCHR1. [(11)C]SNAP-7941 is a carbon-11 labeled analog of the published MCHR1 antagonist SNAP-7941. The optimum reaction conditions were 2 min reaction time, ≤25°C reaction temperature, and 2 mg/mL precursor (SNAP-acid) in acetonitrile, using [(11)C]CH(3)OTf as methylation agent. [(11)C]SNAP-7941 was prepared in a reliable and feasible manner with high radiochemical yields (2.9±1.6 GBq; 11.5±6.4% EOB, n=15).

Optimization of the radiosynthesis of the Alzheimer tracer 2-(4-N-[11C]methylaminophenyl)-6-hydroxybenzothiazole ([11C]PIB).

[(11)C]PIB is still the standard PET compound for Alzheimer imaging targeting beta-amyloid plaques. We aimed to establish a fully-automated procedure for the synthesis and purification of [(11)C]PIB with a high degree of reliability and improved specific activity as well as a suitable and fast quality control assay. The optimum reaction conditions were 75°C, 4 mg/mL precursor yielding at 48.0±2.7% (EOS, based on [(11)C]CH(3)OTf, corrected for decay), 183±14 GBq/µmol specific activity and >99% radiochemical purity. Time consumption was kept to a minimum (40 min from EOB) and overall yields were enough to serve 2 consecutive patients with a single preparation.

"Label and go"--a fast and easy radiolabelling method for pellets.

For the development and process optimization of pharmaceutical equipment, it is important to investigate the underlying processes. Taking the fluidized bed technology as an example, the study of particle flow pattern and convection of the particles within the functional unity is essential for construction and process improvement. With positron emission particle tracking (PEPT) it is possible to study the real-time particle motion with radiolabelled particles. We established a fast and simple labelling technique with [(18)F]fluoride for pellets composed of Avicel and anion exchange resin. The uptake of activity ranged from 1.3% to 1.7% per mg and 8.6% to 16.3% per pellet. A specific binding of [(18)F]fluoride with increasing degree of anion exchange resin in the pellets could be observed.

Lectin binding patterns reflect the phenotypic status of in vitro chondrocyte models.

In vitro studies using chondrocyte cell cultures have increased our understanding of cartilage physiology and the altered chondrocytic cell phenotype in joint diseases. Beside the use of primary cells isolated from cartilage specimens of donors, immortalized chondrocyte cell lines such as C-28/I2 and T/C-28a2 have facilitated reproducible and standardized experiments. Although carbohydrate structures appear of significance for cartilage function, the contribution of the chondrocyte glycocalyx to matrix assembly and alterations of the chondrocyte phenotype is poorly understood. Therefore, the present study aimed to evaluate the glycoprofile of primary human chondrocytes as well as of C-28/I2 and T/C-28a2 cells in culture. First, the chondrocytic phenotype of primary and immortalized cells was assessed using real-time reverse transcriptase polymerase chain reaction, immunofluorescence, and glycosaminoglycans staining. Then, a panel of lectins was selected to probe for a range of oligosaccharide sequences determining specific products of the O-glycosylation and N-glycosylation pathways. We found that differences in the molecular phenotype between primary chondrocytes and the immortalized chondrocyte cell models C-28/I2 and T/C-28a2 are reflected in the glycoprofile of the cells. In this regard, the glycocalyx of immortalized chondrocytes was characterized by reduced levels of high-mannose type and sialic acid-capped N-glycans as well as increased fucosylated O-glycosylation products. In summary, the present report emphasizes the glycophenotype as an integral part of the chondrocyte phenotype and points at a significant role of the glycophenotype in chondrocyte differentiation.