Role of structural variations of polysaccharide antigens in the pathogenicity of Gram-negative bacteria.

Pathogenic bacteria employ many strategies to overcome the host immune system for extended survival and propagation in their hosts. Components of the bacterial outer-membrane play an important role in this process. When invading the host, Gram-negative bacteria often use a strategy, known as phase variation, that involves a reversible change in antigenic determinants, frequently polysaccharides. This means that the genes encoding the outer-membrane antigens undergo reversible changes within repeated simple DNA sequence motifs. The antigenic structure of the bacterial outer-membrane is influenced by the character of the host immune system, as well as by the targets for bacterial invasion. When the selection pressure of the immune system is absent or weak, bacteria can fail to synthesise the outer-membrane antigens, which are not needed at that time. Smooth-to-rough (S-R) mutation, an economical and often irreversible process in some Gram-negative bacteria, involves the gradual shortening of the lipopolysaccharide (LPS) O-chain. Under certain conditions, e.g., propagation in embryonated eggs or cell lines, some bacteria will cease synthesis of the complete LPS O-chain because it is an energy-demanding process. A type of gradual shortening of the LPS O-chain by Coxiella burnetii, traditionally called phase variation, is used in serological tests for the diagnosis of Q fever. This review discusses the role and function of polysaccharides, especially LPS produced by some Gram-negative bacteria, in bacterial survival.

Effect of pressure on the magnetic properties of TM(3)[Cr(CN)(6)](2)·12H(2)O.

We present the results of magnetization and AC susceptibility measurements performed on ferrimagnetic Mn(3)(2+)[Cr(III)(CN)(6)](2)·12H(2)O and ferromagnetic Ni(3)(2+)[Cr(III)(CN)(6)](2)·12H(2)O systems under pressures up to 0.9 GPa in a commercial SQUID magnetometer. The magnetization process is affected by pressure: magnetization saturates at higher magnetic field, saturated magnetization µ(s) of Ni(3)[Cr(CN)(6)](2) is reduced and almost unaffected for Mn(3)[Cr(CN)(6)](2) at low temperatures. The Curie temperature T(C) of Mn(3)[Cr(CN)(6)](2) increases with the applied pressure, ΔT(C)/Δp = 25.5 K GPa(-1), due to a strengthened super-exchange antiferromagnetic interaction J(AF), but it is not affected significantly in the case of Ni(3)[Cr(CN)(6)](2) with a dominant ferromagnetic J(F) super-exchange interaction. The increase in the J(AF) interaction is attributed to the enhanced value of the single electron overlapping integral S and the energy gap Δ of the mixed molecular orbitals t(2g) (Mn(2+)) and t(2g) (Cr(III)) induced by pressure.

Detergent extract from chlamydial elementary bodies used as antigen for enzyme-linked immunosorbent assay.

Soluble antigen (SA) from chlamydial elementary bodies (EBs) was extracted with N-lauroylsarcosine. The extracted SA composed of lipopolysaccharide (LPS) and proteins was compared with EBs using an enzyme-linked immunosorbent assay (ELISA). Patient sera from natural chlamydial infections exhibited ELISA mean absorbance (A(492) and A(405/650)) values 2-5 times higher with SA than with EBs, resulting in a better discrimination between positive and negative human sera.

Phase variation of lipopolysaccharide of Coxiella burnetii, strain Priscilla during chick embryo yolk sac passaging.

Changes of lipopolysaccharide (LPS) of Coxiella burnetii strain Priscilla during chick embryo yolk sac passaging were observed by SDS-PAGE and immunoblot analysis. The course of LPS phase variation was similar to that found in other C. burnetti strains, i.e. a conversion of the phase I to the intermediate phase II after 10 passages. The intermediate phase II LPS of Priscilla strain was also detectable by immunoblot analysis using immune serum against Priscilla strain in the 30th passage.

Green fluorescent protein as a detection marker for Coxiella burnetii transformation.

The molecular biological study of the obligate intracellular bacterium Coxiella burnetii is hampered because of the lack of an efficient DNA transformation system. We used expression of the green fluorescent protein (GFP) in addition to ampicillin resistance as a selection marker for detection of transformed C. burnetii cells. Fluorescent microscopy studies revealed that transformed C. burnetii cells can be detected easily inside the host cell line. A high level of GFP expression was reached with the strong Escherichia coli trc (trp/lac) promoter. The use of GFP not only provides a convenient marker for transformation of C. burnetii, but also allows detection of this obligate intracellular pathogen inside host eukaryotic cells. Possible applications for GFP in the study of host-pathogen interactions are discussed.

Electron-microscopic study of the effect of various extractants on the morphology of Coxiella burnetii.

This paper presents the results of the effect of trichloroacetic acid (TCA) at two different temperatures and the effect of a mixture of detergents (D) on Coxiella burnetii. Both TCA and D caused a large destruction of C. burnetii cells. In both cases complexes of high-molar-mass components from the outer membrane were extracted. In the D case not only proteins but a mixture with other high-molar-mass structures were released from the destroyed cells. By extraction of TCA, the antigenic complex composed of lipopolysaccharides (LPS), proteins and phospholipids was released. The effect of the D mixture on C. burnetii causes their complete destruction. The TCA causes a surface destruction of cells but it does not cause complete disintegration. The small-cell variants (SCV) in both cases were shown to be more stable compared to the large-cell variants (LCV).

Cross-reactivity between Coxiella burnetii and chlamydiae.

A cross-reactivity among some strains of Coxiella burnetii and chlamydiae with immune rabbit and mouse sera in ELISA and immunoblot analysis was observed. In the latter, the cross-reactivity disappeared after a treatment of C. burnetii or C. psittaci with proteinase K, which indicates that only proteins were involved. The observed cross-reactivity was not influenced by host chick embryo yolk sac proteins. After adsorption of immune rabbit sera with homologous corpuscular antigens the cross-reactivity disappeared. The possibility of influence of such cross-reactivity on serological diagnosis of C. burnetii or chlamydiae infections is discussed.

Kinetics of phospholipid extraction from Coxiella burnetii.

Kinetics of phospholipid extraction from purified suspensions of Coxiella burnetii in phase I by various chloroform--methanol mixtures at various temperatures were evaluated based on the amount of phosphorus extracted. Extraction by a boiling (53 degrees C) 2:1 chloroform:methanol mixture proved to be the most efficient.

Improved method of isolation of Coxiella burnetii proteins.

An improved method of isolation of Coxiella burnetti proteins was developed. It consists of a combination of detergent (sodium dodecyl sulphate (SDS) or sodium deoxycholate (DOC) and hot phenol treatments. The resulting phenol phase (PP) contained either lipopolysaccharide-(LPS) free proteins (DOC extraction) or proteins contaminated with LPS (SDS extraction), while the water phase (WP) contained LPS. Isolated C. burnetii proteins induced in mice and rabbits antibodies reacting in immunoblot analysis with both phase I and II C. burnetii corpuscles. A rabbit serum against C. burnetii prepared by DOC-phenol extraction did not react with purified I C. burnetii LPS in immunoblot analysis.

Immunological consequences of Coxiella burnetii phase variation.

The influence of the number of passages in chick embryo yolk sac (EPs) on the properties of the lipopolysaccharide (LPS) and other antigens of Coxiella burnetii Priscilla strain in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE), immunoblot analysis, enzyme-linked immunosorbent assay (ELISA) and complement-fixation reaction (CFR) test has been studied. Three phases in the phase variation of Coxiella burnetii could be distinguished by these methods: phase I lasting up to the 20th passage (EP 20), intermediate phase corresponding to EP 20-EP 70, and phase II beginning at EP 80. The changes in LPS were more marked than those in proteins which conserved their immunoblot profile up to EP 80. The phase II was clearly demonstrated by all the methods used.

Coxiella burnetii phase I and II proteins studied by SDS-page.

Coxiella burnetii cells in both phase I and II reveal in sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) similar protein profiles with only small differences. C. burnetii protein profile in SDS-PAGE depended on the method of purification of C. burnetii cells from chick embryo yolk sacs. Immune mouse sera against C. burnetii phase I cells recognized in phase I and II cell protein profiles mainly the 61 K and 29 K proteins by the immunoblot method. Hyperimmune mouse and rabbit sera against phase I and II cells reacted in different way with phase I and II cells. Sera against phase I cells recognized in both phase I and II profiles more protein bands than sera against phase II cells. Thus phase I LPS present in phase I cells exerted adjuvant effect on the antibody response in animals immunized with phase I cells.

Chemical composition of phase I Coxiella burnetii soluble antigen prepared by trichloroacetic acid extraction.

Optimal conditions of extraction (time and temperature) by trichloroacetic acid of soluble antigen from phase I Coxiella burnetii (TCAE), possessing protective properties and used as a chemovaccine against Q fever in men, were studied. Extracts prepared under various conditions were analysed for their polysaccharide, protein and phosphorus contents. Forty-five min of extraction at 0 degrees C were sufficient to obtain a soluble antigen reacting in immunodiffusion with hyperimmune rabbit antiserum. The polysaccharide contents decreased with prolonged extraction at 0 degrees C. At higher extraction temperatures (37 and 100 degrees C), the polysaccharide contents increased while that of proteins decreased. TCAE prepared at 100 degrees C gave no positive immunodiffusion reaction.

Immunogenicity of Coxiella burnetii whole cells and their outer membrane components.

The immunogenicity and protective efficacy of the phase I and phase II Coxiella burnetii whole cells (Cb I and Cb II) and their outer membrane components (OMC), i.e. phase I trichloroacetic acid extract (TCAE), phase I 29 K protein (PRO), phase I and II lipopolysaccharides (LPS I, LPS II), polysaccharides (PS I, PS II), and lipid A (LA I, LA II), were compared. The highest immune response was observed in BALB/c mice by Cb I in both humoral immunity and lymphocyte transformation assays, and in the protective effect as well. The immune response was also significant by Cb II, but their protective capacity was low. The OMC reacted variously. Only TCAE and PRO gave a high value of humoral immunity evaluated by the serological methods. All OMC reacted in the haemolytic plaque assay giving different responses. Lymphoproliferation of splenocytes was positive with all OMC using both Cb I and Cb II antigens with the exception of PS I and PS II in the case of Cb II antigen. The induction of protection against infectious Cb I was demonstrated after immunization with TCAE, PRO, and LPS I. Other OMC did not induce protection against this agent.

Biological properties of chloroform/methanol extracts of Coxiella Burnetii.

Biological properties of antigens of whole cells of C. burnetii in phase I (WCI), their residue (CMR) and extract (CME) obtained by extraction of C. burnetti with chloroform/methanol mixture were subjected to a comparative study. It was shown that CME is able to react specifically with sera containing antibodies against WCI. The inoculation of animals with CME, but not with WCI and CMR does not cause changes of humoral and cell immunity to Coxiella and does not strengthen nonspecific antibacterial activity, but it can stimulate the formation of antibodies. Morphological changes of internal organs of mice were most expressed after the CMR inoculation and were practically absent after the CME inoculation.

[Detection and diagnosis of hereditary monogenic neurologic diseases in Slovakia]. / Záchytnost a diagnostika dedicných monogénne podmienených neurologických ochorení na Slovensku.

Authors present a clinical symptoms recapitulation of the most important monogenic hereditary neuromuscular diseases, their molecular-genetic causes and the possibilities of diagnostic on the level of DNA analysis. Low detectability of these pathologic states in Slovak republic is stressed and possible causes of this state are analyzed. (Ref. 10.)

[Indirect diagnosis of type I neurofibromatosis using RFLP in Slovak families]. / Nepriama diagnostika neurofibromatózy typ 1 pomocou RFLP v slovenských rodinách.

Neurofibromatosis type I clinical diagnosis confirmation as well as antenatal diagnostics of the disease are recently provided by molecular genetics. The authors analyze 17 Slovak families with multiple NFI incidence, in whom the detection of mutated gene transfer was performed using indirect diagnostics-bound with of restrictive fragments length polymorphism RFLP. With the help of PCR 7 polymorphic sequencies were amplified and subsequently broken with restrictive endonucleases localized close to the neurofibrin gene. The system informative capacity was comparable with the results of other Caucasian population studies. Although direct detection of mutation is the perspective of the diagnostics, binding analysis in informative families with multiple incidence of the disease provides reliable and cheaper possibility of NFI diagnostic on the level of DNA analysis. (Tab. 1, Fig. 3, Ref. 26.)

[Chromosome aberrations in children with the Williams-Beuren syndrome]. / Chromozómové aberácie u detí s Williamsovým-Beurenovým syndrómom.

Karyotypic examinations were carried out in 10 children suffering from Williams-Beuren's syndrome. Chromosomal aberrations were established in two of the children. In one case the mosaic pattern of Klinefelter's syndrome was recorded, evidently presenting an instance of coincidence. In the other case deletion of the long arm of chromosome 6 (q22.2q23) was demonstrated, which according to the authors' hypothesis may represent an alternative localization of the phenotypic traits of Williams-Beuren's syndrome or of its assumed underlying defect, namely derangement of the regulation of calcium metabolism. The value of cytogenetic examination of children with Williams-Beuren's syndrome in elucidating the so far obscure pathogenesis of this disease is being emphasized.

[Presence of Y-specific sequences in patients with Turner syndrome as a presymptomatic marker of increased risk of gonadoblastoma]. / Prítomnost Y-specifických sekvencií u pacientok s Turnerovým syndrómom ako presymptomatický marker zvýseného rizika gonadoblastómu.

The risk of the origin of neoplasms in patients with gonadal dysgenesis and the presence of Y chromosome mosaicism has been known for a long period. The majority of hidden mosaicism is however not detectable by means of cytogenetic methods. The authors of this study deal with the detection of Y specific chromosomal sequences in 86 patients with Turner syndrome by means of polymerase chain reaction (PCR) and compare the results of this method with cytogenetic findings. The presence of Y specific sequences was proven in 8 patients (9.3%) which correlates with the results of several recent studies. In two cases, the Y chromosome fragment was verified also cytogenetically, in five patients, the diagnose was made more accurate at an originally non-specified marker, and in two cases, the cytogenetic examination has assessed the finding of X chromosome only. PCR is a more sensitive and a more precise method of the assessment of Y chromosome mosaicism in patients with Turner syndrome enabling more effectively to single out persons under the risk of rudimentary gonads gonadoblastoma development. (Fig. 5, Ref. 32.)