Role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis. II. Antibodies to Salmonella and Yersinia in sera and synovial fluids

IgA, IgG and IgM antibodies against Yersinia Yop proteins, Yersinia LPS and Salmonella LPS from different serogroups were determined by enzyme-linked immunosorbent assay (ELISA) in a 885 serum samples and 92 synovial fluids. The control group consisted of 200 healthy blood donors. Compared with control subjects, patients with arthritis showed significantly increased titres of antibodies against Yersinia Yop, Yersinia LPS and Salmonella LPS appropriately in 21.7%, 44.0% and 56.0% serum samples. The prevalence of positive antibody levels was highest in Yersinia serogroup O3 and Salmonella serogroup B and D antibodies. The IgA titres were found to be much higher in adults than in children and youngsters but IgM titres consequently decreased with age. Investigation of synovial fluids obtained from patients with arthritis showed that Yersinia and Salmonella antibodies in synovial fluid mirror those in serum by concentration, by specificity and by distribution in classes.     

Frequency and susceptibility patterns of non-fermenting gram-negative rods isolated from patients hospitalised in intensive care units of a tertiary care hospital

The aim of the study was to assess frequency and susceptibility to antimicrobial agents of non-fermenting gram-negative rods isolated from clinical specimens obtained from patients requiring intensive care, with emphasis on profile of the unit. Identification of cultured isolates was done using automated VITEK and API systems (bioMerieux, France). Susceptibility to antimicrobial agents was tested by a disk-diffusion method according to the NCCLS recommendations. In total the analysis comprised 425 strains of non-fermenting gram-negative rods, constituting 58.9% of all isolates of gram-negative bacteria. In blood cultures predominated strains of A. baumannii (46.8%) and P. aeruginosa (40.4%), while in cultures of other clinical specimens these bacteria comprised 42.9% and 43.9% of isolates. Major differences were observed in frequency of these species on both ICU units. Strains of non-fermenting rods isolated from blood cultures comprised a lower percentage of strains susceptible to antimicrobials (particularly cefepime and carbapenems) than isolates cultured from other specimens. Strains of A. baumannii resistant to imipenem and meropenem were detected with a frequency of 12.5% and 26.7%, respectively. Resistance of P. aeruginosa strains to carbapenems was 62.2% and 44.3%, respectively. There was a relatively high percentage of strains susceptible to cefepime (82.0%), ceftazidime (78.9%), amikacin (77.8%) and piperacillin/tazobactam (69.7%). Conclusions: 1. There was a predominance (58.9%) of strains of gram-negative non-fermenting rods. 2. Isolates from blood cultures were characterised by a much higher percentage of resistant strains in comparison to other specimens. 3. Strains of A. baumannii resistant to carbapenems were recorded. 4. There were differences in frequency and antimicrobial susceptibility among the strains of P. aeruginosa and A. baumannii depending on the type of clinical specimen and ICU profile.     

Assessing genetic variation to predict the breeding value of winter triticale cultivars and lines

The objective of this study was to evaluate the usefulness of amplified fragment length polymorphism (AFLP) analyses for selection of the best parents for breeding of hybrid winter triticale. Phenotypic diversity was measured for 8 agronomic traits in 10 parents and 27 F1 hybrids. Genotypic diversity was measured by 91 AFLP markers. Coefficients of correlation of genetic similarity (AFLP-GS) with both Euclidean distances and mean values of the traits were generally not significant. A correction of the preliminary binary matrix into trait-specific derived matrices increased the values of 5 correlation coefficients to a significant level. The correlation of AFLP-GS with mid-parent heterosis of grain weight per plant was low but significant (r = -0.452). Our study confirms the effectiveness of marker preselection for obtaining AFLP-GS better correlated with heterosis. The use of derived matrices is promising for reducing the number of cross combinations tested for specific combining ability.     

Interaction of Helicobacter pylori with sialylated carbohydrates: the dependence on different parts of the binding trisaccharide Neu5Ac{alpha}3Gal{beta}4GlcNAc

We have recently shown that binding of Helicobacter pylori to sialylated carbohydrates is dependent on the presence of the carboxyl group and the glycerol chain of Neu5Ac. In this work we investigated the importance of GlcNAc in the binding trisaccharide Neu5Acalpha3Galbeta4GlcNAc and the role of the N-acetamido groups of both Neu5Ac and GlcNAc. An important part of the project was epitope dissection, that is chemical derivatizations of the active carbohydrate followed by binding studies. In addition we used a panel of various unmodified carbohydrate structures in the form of free oligosaccharides or glycolipids. These were tested for binding by hemagglutination inhibition assay, TLC overlay tests, and a new quantitative approach using radiolabeled neoglycoproteins. The studies showed that the N-acetamido group of Neu5Ac is important for binding by H. pylori, whereas the same group of GlcNAc is not. In addition, Fuc attached to GlcNAc, as tested with sialyl-Lewis x, did not affect the binding. Free Neu5Ac was inactive as inhibitor, and Neu5Acalpha3Gal turned out to be active. The binding preference for neolacto structures was confirmed, although one strain also was inhibited by lacto chains. The combined results revealed that an intact Neu5Ac is crucial for the interactions with H. pylori. Parts of Gal also seem to be necessary, whereas the role of the GlcNAc is secondary. GlcNAc does influence binding, however, primarily serving as a guiding carrier for the binding epitope rather than being a part of the binding structure.     

Assessment of potential application of binary mixtures of 2,4-d with novel aminophosphonates

A series of new aminoalkane- and aminofluorenephosphonates was synthesized for agrochemical application. The particular compounds had different alkyl substituents at the carbon, nitrogen and phosphorus atoms. Their pesticidal activity was checked by applying various experimental methods. These included the measurements of compounds' potency: to inhibit growth of cucumber and germination of white mustard seeds, to influence on the membrane potential of algae and to damage human erythrocyte membranes resulting in hemolysis. All the aminophosphonates were also used in equimolar binary mixtures with the well-known herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), to check, if using such mixtures, the biological efficiencies found for particular compounds could be enhanced due to interactions between aminophosphonates and 2,4-D. The results demonstrated, that depending on the structural features of the compounds, the final effects differed from antagonistic, through additive to the most promising synergistic ones. However, the type of interaction between 2,4-D and the compounds studied found in different experiments was somewhat different. In order to estimate those effects various statistical methods were used (toxic unit method, isobole method).     

Tanshinone production in roots of micropropagated Salvia przewalskii Maxim

Production of tanshinones (tanshinone I and IIA) was determined in roots of Salvia przewalskii micropropagated plants. It was found that the total tanshinone content (tashinone I and tashinone IIA) was dependent on the age of the analyzed plants. The roots of 2-year-old in vitro regenerated plants at flowering stage produced highest tanshinone levels (3.8 mg/g dry weight of tanshinone I and 7.6 mg/g dry weight of tanshinone IIA).     

Age-related increase of kynurenic acid in human cerebrospinal fluid - IgG and beta2-microglobulin changes

Kynurenic acid (KYNA) is an endogenous metabolite in the kynurenine pathway of tryptophan degradation and is an antagonist at the glycine site of the N-methyl-D-aspartate as well as at the alpha 7 nicotinic cholinergic receptors. In the brain tissue KYNA is synthesised from L-kynurenine by kynurenine aminotransferases (KAT) I and II. A host of immune mediators influence tryptophan degradation. In the present study, the levels of KYNA in cerebrospinal fluid (CSF) and serum in a group of human subjects aged between 25 and 74 years were determined by using a high performance liquid chromatography method. In CSF and serum KAT I and II activities were investigated by radioenzymatic assay, and the levels of beta(2)-microglobulin, a marker for cellular immune activation, were determined by ELISA. The correlations between neurochemical and biological parameters were evaluated. Two subject groups with significantly different ages, i.e. <50 years and >50 years, p < 0.001, showed statistically significantly different CSF KYNA levels, i.e. 2.84 +/- 0.16 fmol/microl vs. 4.09 +/- 0.14 fmol/microl, p < 0.001, respectively; but this difference was not seen in serum samples. Interestingly, KYNA is synthesised in CSF principally by KAT I and not KAT II, however no relationship was found between enzyme activity and ageing. A positive relationship between CSF KYNA levels and age of subjects indicates a 95% probability of elevated CSF KYNA with ageing (R = 0.6639, p = 0.0001). KYNA levels significantly correlated with IgG and beta(2)-microglobulin levels (R = 0.5244, p = 0.0049; R = 0.4253, p = 0.043, respectively). No correlation was found between other biological parameters in CSF or serum. In summary, a positive relationship between the CSF KYNA level and ageing was found, and the data would suggest age-dependent increase of kynurenine metabolism in the CNS. An enhancement of CSF IgG and beta(2)-microglobulin levels would suggest an activation of the immune system during ageing. Increased KYNA metabolism may be involved in the hypofunction of the glutamatergic and/or nicotinic cholinergic neurotransmission in the ageing CNS.     

Structural and serological characterization of the major glycolipid from Rothia mucilaginosa

Structural studies on the major glycolipid isolated from Rothia mucilaginosa were carried out utilising specific chemical degradation, NMR spectroscopy and matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI TOF-MS). The glycolipid was found to be a dimannosylacylmonoglyceride in which the carbohydrate part was the glycerol-linked dimannoside alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-sn-Gro (Man A-Man B-Gro), of which Man B was esterified at O-6 by a fatty acid residue. A second fatty acid substituted the secondary methylene position of the glycerol residue, in contrast to the glycolipid previously found in R. dentocariosa and Saccharopolyspora strains, in which the second fatty acid esterified the primary methylene position of glycerol. Results of the ELISA experiment with rabbit specific antibacterial sera indicate that these two major glycolipids are antigenic, and the patterns of serological reactivity are similar but not identical.     

Nitric oxide and platelet energy metabolism

This study was undertaken to determine whether nitric oxide (NO) can affect platelet responses through the inhibition of energy production. It was found that NO donors: S-nitroso-N-acetylpenicyllamine, SNAP, (5-50 microM) and sodium nitroprusside, SNP, (5-100 microM) inhibited collagen- and ADP-induced aggregation of porcine platelets. The corresponding IC50 values for SNAP and SNP varied from 5 to 30 microM and from 9 to 75 microM, respectively. Collagen- and thrombin-induced platelet secretion was inhibited by SNAP (IC50 = 50 microM) and by SNP (IC50 = 100 microM). SNAP (20-100 microM), SNP (10-200 microM) and collagen (20 microg/ml) stimulated glycolysis in intact platelets. The degree of glycolysis stimulation exerted by NO donors was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or uncouplers (2,4-dinitrophenol). Neither the NO donors nor the respiratory chain blockers affected glycolysis in platelet homogenate. SNAP (20-100 microM) and SNP (50-200 microM) inhibited oxygen consumption by platelets. The effect of SNP and SNAP on glycolysis and respiration was not reduced by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-stimulated guanylate cyclase. SNAP (5-100 microM) and SNP (10-300 microM) inhibited the activity of platelet cytochrome oxidase and had no effect on NADH:ubiquinone oxidoreductase and succinate dehydrogenase. Blocking of the mitochondrial energy production by antimycin A slightly affected collagen-evoked aggregation and strongly inhibited platelet secretion. The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.     

Monomeric recombinant TCR ligand reduces relapse rate and severity of experimental autoimmune encephalomyelitis in SJL/J mice through cytokine switch

Our previous studies demonstrated that oligomeric recombinant TCR ligands (RTL) can treat clinical signs of experimental autoimmune encephalomyelitis (EAE) and induce long-term T cell tolerance against encephalitogenic peptides. In the current study, we produced a monomeric I-A(s)/PLP 139-151 peptide construct (RTL401) suitable for use in SJL/J mice that develop relapsing disease after injection of PLP 139-151 peptide in CFA. RTL401 given i.v. or s.c. but not empty RTL400 or free PLP 139-151 peptide prevented relapses and significantly reduced clinical severity of EAE induced by PLP 139-151 peptide in SJL/J or (C57BL/6 x SJL)F(1) mice, but did not inhibit EAE induced by PLP 178-191 or MBP 84-104 peptides in SJL/J mice, or MOG 35-55 peptide in (C57BL/6 x SJL/J)F(1) mice. RTL treatment of EAE caused stable or enhanced T cell proliferation and secretion of IL-10 in the periphery, but reduced secretion of inflammatory cytokines and chemokines. In CNS, there was a modest reduction of inflammatory cells, reduced expression of very late activation Ag-4, lymphocyte function-associated Ag-1, and inflammatory cytokines, chemokines, and chemokine receptors, but enhanced expression of Th2-related factors, IL-10, TGF-beta3, and CCR3. These results suggest that monomeric RTL therapy induces a cytokine switch that curbs the encephalitogenic potential of PLP 139-151-specific T cells without fully preventing their entry into CNS, wherein they reduce the severity of inflammation. This mechanism differs from that observed using oligomeric RTL therapy in other EAE models. These results strongly support the clinical application of this novel class of peptide/MHC class II constructs in patients with multiple sclerosis who have focused T cell responses to known encephalitogenic myelin peptides.