Bone mineral density changes of lumbar spine and femur in osteoporotic patient treated with bisphosphonates and beta-hydroxy-beta-methylbutyrate (HMB): Case report.

RATIONALE: Currently available approaches to osteoporosis treatment include application of antiresorptive and anabolic agents influencing bone tissue metabolism. The aim of the study was to present bone mineral density (BMD) changes of lumbar spine in osteoporotic patient treated with bisphosphonates such as ibandronic acid and pamidronic acid, and beta-hydroxy-beta-methylbutyrate (HMB). PATIENT CONCERNS: BMD and volumetric BMD (vBMD) of lumbar spine were measured during the 6 year observation period with the use of dual-energy X-ray absorptiometry (DEXA) and quantitative computed tomography (QCT). DIAGNOSES: The described case report of osteoporotic patient with family history of severe osteoporosis has shown site-dependent response of bone tissue to antiosteoporotic treatment with bisphosphonates. INTERVENTIONS AND OUTCOMES: Twenty-five-month treatment with ibandronic acid improved proximal femur BMD with relatively poor effects on lumbar spine BMD. Over 15-month therapy with pamidronic acid was effective to improve lumbar spine BMD, while in the proximal femur the treatment was not effective. A total of 61-week long oral administration with calcium salt of HMB improved vBMD of lumbar spine in the trabecular and cortical bone compartments when monitored by QCT. Positive effects of nearly 2.5 year HMB treatment on BMD of lumbar spine and femur in the patient were also confirmed using DEXA method. LESSONS: The results obtained indicate that HMB may be applied for the effective treatment of osteoporosis in humans. Further studies on wider human population are recommended to evaluate mechanisms influencing bone tissue metabolism by HMB.

Free-Living Species of Carnivorous Mammals in Poland: Red Fox, Beech Marten, and Raccoon as a Potential Reservoir of Salmonella, Yersinia, Listeria spp. and Coagulase-Positive Staphylococcus.

The objective of the study was to examine a population of free-living carnivorous mammals most commonly found in Poland (red fox, beech marten, and raccoon) for the occurrence of bacteria that are potentially pathogenic for humans and other animal species and to determine their virulence potential (the presence of selected virulence genes). From the total pool of isolates obtained (n = 328), we selected 90 belonging to species that pose the greatest potential threat to human health: Salmonella spp. (n = 19; 4.51%), Yersinia enterocolitica (n = 10; 2.37%), Listeria monocytogenes and L. ivanovii (n = 21), and Staphylococcus aureus (n = 40; 9.5%). The Salmonella spp. isolates represented three different subspecies; S. enterica subsp. enterica accounted for a significant proportion (15/19), and most of the serotypes isolated (S. Typhimurium, S. Infantis, S. Newport and S. Enteritidis) were among the 10 non-typhoidal Salmonella serotypes that are most often responsible for infections in Europe, including Poland. Y. enterococlitica was detected in the smallest percentage of animals, but 60% of strains among the isolates tested possessed the ail gene, which is responsible for attachment and invasion. Potentially pathogenic Listeria species were isolated from approx. 5% of the animals. The presence of all tested virulence genes was shown in 35% of L. monocytogenes strains, while in the case of the other strains, the genes occurred in varying numbers and configurations. The presence of the inlA, inlC, hlyA, and iap genes was noted in all strains, whereas the genes encoding PI-PLC, actin, and internalin Imo2821 were present in varying percentages (from 80% to 55%). S. aureus was obtained from 40 individuals. Most isolates possessed the hla, hld (95% for each), and hlb (32.5%) genes encoding hemolysins as well as the gene encoding leukotoxin lukED (70%). In a similar percentage of strains (77.5%), the presence of at least one gene encoding enterotoxin was found, with 12.5% exhibiting the presence of egc-like variants. In two animals, we also noted the gene encoding the TSST-1 toxin. The results of the study showed that free-living animals may be a significant reservoir of bacteria that are potentially pathogenic for humans. The results of the statistical analysis revealed that, among the animals species studied, the red fox constitutes the most important source of infections.

Investigation of the composition and antibacterial activity of Ukrain™ drug using liquid chromatography techniques.

The greater celandine (Chelidonium majus L.) has been known for the centuries as a medicinal plant. One of the therapeutic agents based on C. majus is anticancer drug Ukrain™ known as a semi-synthetic C. majus alkaloid derivative. Although there are no doubts about antitumor properties of the drug, there is still controversy about its composition. In this study, Ukrain™ was subjected to TLC and LC-MS/MS analyses to compare it with C. majus alkaloid root extract and to determine its composition. Moreover, microbiological activity of both Ukrain™ and the alkaloid extract were tested against Bacillus subtilis strains using TLC-direct bioautography. Sanguinarine, chelidonine, α-homochelidonie and chelerythrine were found to have antibacterial properties. Besides chelidonine, sanguinarine, chelerythrine, protopine, allocryptopine, homochelidonie, berberine and coptisine reported earlier in literature, the presence of stylopine, norchelidonine, dihydrochelidonine and hydroberberine in Ukrain™ was detected, and here they have been reported for the first time.

A Comparison of Antibacterial Activity of Selected Thyme (Thymus) Species by Means of the Dot Blot Test with Direct Bioautographic Detection.

Bioautography carried out with the aid of thin-layer chromatographic adsorbents can be used to assess antibacterial activity in samples of different origin. It can either be used as a simple and cost-effective detection method applied to a developed chromatogram, or to the dot blot test performed on a chromatographic plate, where total antibacterial activity of a sample is scrutinized. It was an aim of this study to compare antibacterial activity of 18 thyme (Thymus) specimens and species (originating from the same gardening plot and harvested in the same period of time) by means of a dot blot test with direct bioautography. A two-step extraction of herbal material was applied, and at step two the polar fraction of secondary metabolites was obtained under the earlier optimized extraction conditions [methanol-water (27+73, v/v), 130°C]. This fraction was then tested for its antibacterial activity against Bacillus subtilis bacteria. It was established that all investigated extracts exhibited antibacterial activity, yet distinct differences were perceived in the size of the bacterial growth inhibition zones among the compared thyme species. Based on the results obtained, T. citriodorus "golden dwarf" (sample No. 5) and T. marschallianus (sample No. 6) were selected as promising targets for further investigations and possible inclusion in a herbal pharmacopeia, which is an essential scientific novelty of this study.

Use of different cell lines for in vitro cultures of bovine respiratory syncytial virus.

This study compared the use of different cell lines for in vitro cultures of bovine respiratory syncytial virus (BRSV). The BRSV 375 strain and 3 nasal swabs obtained from Simmental calves were used for this study. The culture was performed on 3 cell lines: bovine kidney cells (LLC-PK1), bovine tracheal cells (TBTR) and primary chicken embryo-related cells (CER). A comparative analysis of titres was performed using a microplate agglutination test with human group O erythrocytes and bovine erythrocytes. The presence of BRSV in all cell lines was confirmed using the reverse transcriptase polymerase chain reaction (RT-PCR) method. The first small refractile changes in the LLC-PK1 cells occurred at 48h after infection. Syncytial changes were noted 4 days after incubation. Large refractile cell changes were observed on day 3 of growth in the TBTR culture. Syncytia were observed on the second day after infection in subsequent passages. The cytopathic effect in the CER cells occurred 24h after infection, and syncytia appeared after 3 passages. Changes in syncytia indicate an adaptation of the virus for the infection of cells other than tracheal cells in primary and secondary cultures. The highest viral titre was obtained using the TBTR line. The titres obtained in the LLC-PK1 and CER cultures averaged 10(1.86)/ml. The low virus titres in all culture types suggest the need for research aimed at the optimisation of culture conditions.

Development of a novel direct bioautography-thin-layer chromatography test: optimization of growth conditions for gram-positive bacteria, Bacillus subtilis.

A TLC-direct bioautography (DB) assay using Bacillus subtilis as test bacteria was developed. Various factors affecting the microorganism's viability on the TLC plates were studied and verified for the flumequine standards. The Dhenasar's method called "direct sample determination" was used for TLC; the antibiotic samples were spotted on the TLC plates and subjected to bioautography without developing with a mobile phase. The best preincubation and incubation times of bacterial broth were found to be 1 h at 37 degrees C and 6 h at 37 degrees C. The optimal viscosity of broth was obtained by the addition of agarose to obtain a 0.05% solution in the Mueller-Hinton broth. The best incubation time of seeded TLC plates was 17 h at 37 degrees C. The plates were visualized by spraying with 0.2% aqueous 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide solution and incubated again for 0.5 h at 37 degrees C. The method was validated by determination of linearity, interday and intraday precision, LOD, and LOQ. The calibration curves showed good linearity in the range 0.005-0.5 microg (0.5-50.0 microg/mL). The regression coefficients were 0.9970 and 0.9955 for intraday and interday plots, respectively. The LOD of flumequine equalled 0.5 microg/mL, i.e., 5 ng of the antibiotic in the spot. The sensitivity of the developed TLC-DB test was compared with that of the two most commonly used standard antimicrobial susceptibility assays: agar disc diffusion and agar cylinder diffusion. The obtained minimum inhibitory concentration values clearly indicate much higher sensitivity of the TLC-DB method compared to the standard antimicrobial susceptibility assays.

Applications of novel direct bioautography tests for analysis of antimicrobials: a review.

TLC coupled to direct bioautography detection can be applied to the analysis of various antimicrobial agents in complex matrixes. Because of the lack of commercially available microbiological detection methods, two direct bioautography tests were developed in our laboratory to be used after TLC separation. One method was based on Gram-positive bacteria, Bacillus subtilis, and the other on Gram-negative bacteria, Escherichia coli. These tests can be used for detection and determination of wide spectrum of antimicrobials as well as for other, nontypical purposes, such as choosing the best sample preparation method before the analysis. Some of the more interesting applications of the newly developed tests are described in this paper.

Comparison of deproteinization methods used before TLC-DB and HPLC analysis of flumequine residues in milk.

Seventeen various extraction procedures based on precipitation of proteins in milk samples spiked with flumequine were tested. Several criteria were taken into account, when choosing the most effective. The supernatants were analyzed by thin-layer chromatography direct bioautography (TLC-DB) and high performance liquid chromatography (HPLC-UV). The results obtained from both methods indicate as the best the same deproteinization procedure. The addition of acetonitrile to milk in 1:1 volume proportions gave the highest concentration of flumequine in supernatant and prompt coagulation of proteins in milk samples.

Development of a novel direct bioautography-thin-layer chromatography test: optimization of growth conditions for gram-negative bacteria, Escherichia coli.

With the aim of developing a TLC-direct bioautography assay using Escherichia coli as test bacteria, various parameters influencing the viability of microorganisms on TLC plates were examined and checked for flumequine standards. The optimal times for preincubation and incubation of bacterial broth were 20 h at 37 degrees C and 2 h at 37 degrees C, respectively. The optimal viscosity of the broth was obtained for 0.05% agarose solution in Mueller-Hinton broth. Various incubation times of the seeded TLC plates were also tested (5 h proved to be optimal). After incubation, the plates were sprayed with 0.2% aqueous [3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyltetrazolium bromide] (MTT) solution and incubated for 0.5 h at 37 degrees C. The precision of the method was evaluated by the repeatability (intraday assay) and intermediate precision (interday assay). The regression coefficients were 0.9977 and 0.9968, respectively, for intraday and interday curves. The calibration curves show good linearity in the range of 0.005-0.50 microg (0.5-50.0 microg/mL). The established LOD of flumequine equaled 0.5 microg/mL, i.e., 5 ng flumequine in the spot. The developed direct bioautography test significantly enhances the sensitivity of the TLC method.

Analysis of antimicrobial peptides from porcine neutrophils.

Cationic host defence peptides are important components of innate immunity in pigs and other mammalians. Most of these peptides have a direct antimicrobial activity and they also have a broad spectrum of effects on the host immune system, which may be taken into account in the introduction of novel therapeutics. Our method permits simultaneous isolation of six antibacterial peptides, i.e. prophenin-1, prophenin-2, PR-39, and protegrins 1-3 from a porcine neutrophil crude extract and characterisation of them. Among the obtained peptides the greatest bactericidal activity expressed as MBC was seen in protegrins (10 µg/ml), whereas in the other studied peptides MBC was on the level of 20 µg/ml. Minimal inhibitory concentrations (MIC) reached 10 µg/ml for protegrins 1-3 and 20 µg/ml for prophenins, and PR-39. Within the bactericidal range all isolated peptides didn't show cytotoxicity on cell lines used in our experiment.