In vitro Screening of Essential Oil Active Compounds for Manipulation of Rumen Fermentation and Methane Mitigation.

The objective of this study was to investigate the effects of 11 active compounds of essential oils (ACEO) on rumen fermentation characteristics and methane production. Two trials were conducted. In trial 1, ACEO (eugenol, carvacrol, citral, limonene, 1,4-cineole, p-cymene, linalool, bornyl acetate, α-pinene, and ß-pinene) at a dose of 1,000 µL/L were incubated for 24 h in diluted rumen fluid with a 70:30 forage:concentrate substrate (16.2% crude protein; 36.6% neutral detergent fiber). Three fistulated Holstein cows were used as donors of rumen fluid. The reduction in methane production was observed with nine ACEO (up to 86% reduction) compared with the control (p<0.05). Among these, only limonene, 1,4-cineole, bornyl acetate, and α-pinene did not inhibit volatile fatty acid (VFA) production, and only bornyl acetate produced less methane per mol of VFA compared with the control (p<0.05). In a subsequent trial, the effects on rumen fermentation and methane production of two concentrations (500 and 2,000 µL/L) of bornyl acetate, the most promising ACEO from the first trial, were evaluated using the same in vitro incubation method that was used in the first trial. In trial 2, monensin was used as a positive control. Both doses of bornyl acetate decreased (p<0.05) methane production and did not inhibit VFA production. Positive effects of bornyl acetate on methane and VFA production were more pronounced than the effects of monensin. These results confirm the ability of bornyl acetate to decrease methane production, which may help to improve the efficiency of energy use in the rumen.

Rituximab use in patients with ANCA-associated vasculitis: clinical efficacy and impact on immunological parameters.

Rituximab (RTX) was reported effective in ANCA-associated vasculitis (AAV). We aimed to evaluate clinical efficacy of RTX in AAV along with its impact on immunological parameters. Eighteen RTX-treated AAV patients (M/F 11/7; median age 37.5; 15× PR3-ANCA, 3× MPO-ANCA; 16× relapsing/refractory, 2× first-line therapy) were enrolled. Clinical response, ANCA, total serum IgG levels and cellular immunity parameters were examined. The patients were followed up (FU) for a median of 26 months (range 3-82, 15 for ≥6 months). All patients achieved B cell depletion (lasting 3-24 months). No significant increase was noted in T cell or NK cell subpopulations. At 6 months, partial remission was achieved in 5/15 patients (33 %) and complete in 8 (53 %). The median prednisone dose (30..10 mg/d) and ANCA levels (17.2..2.7 IU/mL) decreased (p < 0.01). RTX retreatment was used in nine (8× pre-emptive, 1× relapse). Six patients relapsed (none of the pre-emptively treated). Three patients died of infection. IgG levels at 3 months decreased compared to baseline (9.0 vs 5.7 g/L, p < 0.01). Higher percentage of HLA-DR+CD3+ cells and lower percentage of CD4+CD45RA+ naive T cells persisted during FU. IFN-γ production increased at 6 months compared to baseline (27.3 vs 41.5 %). No significant change was noted in the intracellular IL-10 and IL-12 production. RTX helped to lower the glucocorticosteroids dose and withdraw cytotoxic drugs in most AAV patients. Hypogammaglobulinaemia was common but well tolerated. Peripheral circulating T cells remained activated despite B cell depletion.

Nucleic acid testing of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus 1, 2 in blood donors in the General University Hospital, Prague.

The Roche Cobas TaqScreen MPX Test v1 - multiplex reverse transcription-real time (MPX RT-Real Time) PCR, performed on Cobas s201 for HCV RNA, HBV DNA, HIV-1 RNA /group M and O/, and HIV-2 RNA was introduced as a supplement to the currently used imunoanalysis method for blood donor´s testing (Abbott CMIA - chemiluminescent microparticle imunoassay, performed on Architect i2000 for anti-HCV, hepatitis B surface antigen (HBsAg)), anti-HIV-1 /group M and O/, anti-HIV-2 and p24 HIV). The results of study could provide valuable arguments to support the discussion about the NAT implementation into the standards of blood donor´s testing in the Czech Republic. Two groups of samples were tested. In the first one, 5074 samples from consecutive blood donors, and in the second one, 5 repository preseroconverted samples from repeat blood donors, who were subsequently confirmed positive for Viral Hepatitis and/or HIV/AIDS by the National Reference Laboratory (NRL), were tested. One sample was found reactive by chemiluminescent microparticle immuno assay (CMIA) and nucleic acid test (NAT) (confirmed HBV-positive in NRL), 31 samples were CMIA-only reactive (15 anti-HCV, 4 HBsAg, 12 anti-HIV/p24, all confirmed negative in NRL) and one pool (6 samples) was found reactive (further individual NAT was negative for all samples) in the first group of samples. One sample was NAT-only reactive (confirmed HCV-positive in NRL) in the second group of samples. Our study confirmed that screening of infectious markers using NAT can reduce the risk of transmitting the monitored infections by blood transfusion in the Czech Republic, even as a country with currently good epidemiological situation.

The frequencies of six important thrombophilic mutations in a population of the Czech Republic.

The primary aim was to determine frequencies of mutations related to risk of venous thrombosis in healthy Caucasians in Central Bohemia. In a cohort of 1527 healthy individuals the frequency of risk alleles for the mutations FV Leiden and FII 20210G>A was 4.5% and 1.3%, respectively. Frequency of 4G PAI-1 allele was 55.5%. Genotype frequencies were: GG 91.03%, GA 8.91%, and AA 0.07% for FV Leiden; GG 97.45%, GA 2.49%, and AA 0.07% for FII 20210G>A; 4G/4G 30.26%, 4G/5G 50.56%, and 5G/5G 19.19% for PAI-1. Frequency of the risk allele A in polymorphism SERPINC1 (IVS +141G >A) was 11.3%, and frequencies of genotypes were as follows: GG 78.36%, GA 20.66%, and AA 0.98%. Frequency of the risk allele T for polymorphism GP6 13254T>C was 87.7%, and frequencies of genotypes were as follows: TT 77.14%, TC 21.15%, and CC 1.70%. Frequency of the risk allele A in polymorphism CYP4V2 (Lys259Gln) was 65.2%, and frequencies of genotypes were: CC 12.25%, CA 45.12 %, and AA 42.63%. All observed genotypes and alleles frequencies were without gender differences. Their occurrences confirm a relatively high prevalence of hereditary thrombophilia predisposition in the Czech Republic.

Endotelial activation and flow-mediated vasodilation in young patients with breast cancer.

Endothelial activation and dysfunction may play a significant role in the progression of breast cancer. In our study we examined markers of endothelial activation (soluble ICAM-1, P-selectin, E-selectin) in 98 young patients with breast cancer (< 40 years). In 50 of them (and 20 age-matched controls) we also measured flow mediated vasodilation. Patients with breast cancer had significantly higher serum levels of soluble E-selectin, P-selectin and ICAM-1, P-selectin was higher in patients with larger tumors, node involvement and seemed to be apredictor of poor outcome. We were unable to find significant difference in the parameters of flow mediated vasodilation between patients with breast cancer and healthy subjects, although both peak blood flow (PBF) and flow mediated vasodilation (FMD) seemed to be skewed compared to healthy subjects toward mean and lower levels. Cluster analysis enabled us to distinguish several larger groups of patients with different degree of endothelial activation and function and different outcome. Group of patients with high E-selectin, high ICAM-1 (higher endothelial activation) and low VEGF (putative endothelial damage) had more frequently negative estrogen receptors and had worse outcome compared to the group of patients with lower E-selectin, lower ICAM-1 and mostly positive estrogen receptors. Further studies of larger groups of patients should help to identify the panel of endothelial markers which could help in predicting the outcome of young patients with breast cancer.

Non-invasive fetal RHD and RHCE genotyping from maternal plasma in alloimmunized pregnancies.

BACKGROUND: In this prospective study, we assessed the feasibility of fetal RH genotyping by analysis of DNA extracted from maternal plasma samples of alloimmunized pregnant women using real-time PCR and primers and probes targeted toward RHD (exon 7 and exon 10) and RHCE (intron 2 and exon 5) genes. METHODS: We analysed 23 alloimmunized pregnant women (16 anti-D, 5 anti-D + C, 2 anti-E) at risk of haemolytic disease of the newborn (HDN) within 11th and 37th week of pregnancy and correlated the results with serological analysis of cord blood. RESULTS AND CONCLUSION: Detection of the presence of the RHD gene, the C and/or E alleles of the RHCE gene in maternal plasma samples is highly accurate and enables implementation in a clinical diagnostic algorithm for following pregnancies at risk for HDN. The absence of RHD gene, the C and/or E alleles of RHCE gene in the current pregnancy excludes the risk of HDN caused by anti-D, anti-C and/or anti-E alloantibodies and the performance of invasive fetal-blood sampling.

Hydrolysis of phytic acid and its availability in rabbits.

Twenty weaned rabbits were fed ad libitum two granulated feeds containing lucerne meal, barley, oats, wheat bran, oilseed meals and sugarbeet pulp in different proportions. Phytate P in these feeds represented 28.6 and 29.3 % of the total P. Digestibility trials were carried out in rabbits 7 and 10 weeks old. Digestibility of phytate P was 82.1 %, on average. Apparent digestibility of total P was 48.1 and 35.5 % in rabbits aged 7 and 10 weeks, respectively. Concentration of P in the faecal DM of these rabbits averaged 11.9 and 14.7 mg/g. Most of the faecal P was phosphates P (68.1 %). Proportion of phytate P in total faecal P was 9.0 %. Age effect on total P digestibility and faecal P concentration was significant (P<0.05). In five in vitro experiments twenty-four rabbits were killed at the age of 11 weeks, digesta samples diluted with physiological saline containing phytic acid and incubated anaerobically. Calculations of phytase activity in segments of the digestive tract were based on the estimation of phytic acid hydrolysed during the first 2 h of incubation. The caecum contained 58.6 % of the phytase activity of the digestive tract. Corresponding relative values for the phytase activity in the stomach, small intestine and colon were 22.3, 7.7 and 11.4 %, respectively. In incubations of the caecal contents, phytic acid was hydrolysed more rapidly at pH 5-6 than in the neutral pH region. The hydrolysis was inhibited by Ca cations, and to a small extent also by phosphate anions. Commercial fungal phytase (Natupho) was highly active in incubations of the contents of the stomach at pH 1.9. It can be concluded that phytic acid is hydrolysed quite efficiently in the digestive tract of rabbits. This hydrolysis occurred mainly in the caecum. Absorption of soluble inorganic phosphates in the gut is incomplete.

Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bifidobacterium pseudolongum.

AIMS: In a rabbit caecal bacterium Bifidobacterium pseudolongum, metabolites of pectin and glucose, and activities of enzymes involved in the degradation of pectin were assayed. Simultaneously, activities of these enzymes were assayed in a rumen pectinolytic strain of Streptococcus bovis. METHODS AND RESULTS: A strain B. pseudolongum P6 which grew best on pectin was selected among bifidobacteria isolated from the rabbit caecum. Cultures of B. pseudolongum P6 grown on pectin produced significantly less formate, lactate and ethanol, and more acetate and succinate than cultures grown on glucose. No CO2 production on pectin was observed. Pectin macromolecule was degraded by extracellular pectinase (EC 3.2.1.15). Cell extracts possessed the activity of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). Streptococcus bovis X4, possessed activity of exopectate lyase and pectinase, but not that of KDPG aldolase. CONCLUSIONS: Our results are consistent with the assumption that in B. pseudolongum P6 acidic products of pectin degradation are catabolized via a modified Entner-Doudoroff pathway, as shown previously in rumen pectin-utilizing bacteria. The missing KDPG aldolase activity in Strep. bovis X4 seems to be the reason for the absence of growth of this bacterium on pectin. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on polysaccharide metabolism in bifidobacteria is fragmentary. This study extends the knowledge on pectin metabolism in intestinal bacteria.

Enrichment of bifidobacteria in the hen caeca by dietary inulin.

Caecal bifidobacterial concentration was increased more than 3-fold in inulin-treated laying hens. The counts of bifidobacteria in birds fed as the control were 9.64, in inulin-diet fed ones 10.17 log CFU/g of caecal content, respectively. Dietary inulin had no effect on caecal microbial metabolite concentration. The proportion of inulin-fermenting bifidobacteria in the total bifidobacteria increased 2-fold in inulin-treated birds.

Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rumen bacterium Lachnospira multiparus.

AIMS: Lachnospira multiparus belongs to the main rumen pectinolytic bacteria. Its carbohydrate metabolism was studied in growth experiments on laboratory fermenters, and using assays of activities of enzymes involved in pectin fermentation. METHODS AND RESULTS: The type strain of this species and two substrates were used. Lachnospira multiparus ATCC 19207 grew on pectin and glucose at a similar rate and had no preference for one or the other substrate. Pectin-grown cultures, however, produced significantly more acetate and less formate, lactate, ethanol, hydrogen, cell dry matter and protein than corresponding cultures grown on glucose. Extracellular exopectate lyase (EC 4.2.2.9) was the principal enzyme degrading the pectin macromolecule. Cell extracts possessed 2-keto-3- deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) and fructosediphosphate aldolase (EC 4.1.2.13) activity. The former enzyme catalyses the final reaction in the Entner-Doudoroff pathway; the latter is the key enzyme of glycolysis and the pentose phosphate pathway. CONCLUSION: These results are consistent with the assumption that acidic products of pectin degradation are catabolized via a modified Entner-Doudoroff pathway. Phosphogluconate was not metabolized by cell extracts of the strain studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This suggests that the conventional Entner-Doudoroff pathway of glucose utilization does not operate in this bacterium, presumably because of the lack of 6-phosphogluconate dehydrase (EC 4.2.1.12) activity.

Stoichiometry of glucose and xylose fermentation in Butyrivibrio fibrisolvens 787.

Glucose and xylose are the principal monomeric units of plant carbohydrates. Butyrivibrio fibrisolvens, an important rumen bacterium, converted both sugars to formate, acetate, butyrate and lactate. More metabolites and less cell biomass was formed from xylose than from glucose. In cultures with both substrates, glucose was utilized preferentially. Growth on glucose was more rapid than on xylose. No phosphoketolase activity (EC 4.1.2.9) was detected in this strain. More carbohydrate and less protein was found in glucose-grown cells than in cells grown on xylose. Growth yields of protein were higher on xylose, in spite of the fact, that yields of cellular dry matter were higher on glucose.