Vaccine model of antiphospholipid syndrome induced by tetanus vaccine.

Successful induction of antiphospholipid syndrome (APS) in two different non-autoimmune prone mouse strains, BALB/c and C57BL/6, was achieved by tetanus toxoid (TTd) hyperimmunization using different adjuvants (glycerol or aluminium hydroxide), and different adjuvant pretreatments (glycerol or Complete Freund's Adjuvant (CFA)). APS had different manifestations of reproductive pathology in BALB/c and C57BL/6 mice: fetal resorption (as a consequence of extreme T-cell activation obtained in the course of pretreatment), and lowering of fecundity (as a consequence of polyclonal B-cell stimulation), respectively. In BALB/c mice fetal resorption coincided with glycerol and CFA pretreatments, while in C57BL/6 mice lowering of fecundity was most obvious in CFA-pretreated mice immunized with TTd in aluminium hydroxide. Both molecular mimicry and polyclonal B-cell activation occur in APS induction, with molecular mimicry effects being dominant in BALB/c mice, and polyclonal cell activation being dominant in C57BL/6 mice. Confirmation of molecular mimicry effects, which in the condition of T-cell stimulation generated fetal resorptions in the BALB/c strain, was achieved by passive infusion of monoclonal antibody (MoAb) T-26 specific for TTd and anti-ß(2)-glycoprotein I obtained after TTd hyperimunization. High polyclonal B-cell activation in C57BL/6 mice prevented fetal resorption but induced fecundity lowering, as was the case in passive administration of MoAb T-26 in this mouse strain. Passive infusion of anti-idiotypic MoAb Y7 into C57BL/6 mice induced fetal resorptions and confirmed the above suggestion on the protective role of polyclonal B-cell stimulation in fetal resorptions.

The identification of a low molecular mass bacteriocin, rhamnosin A, produced by Lactobacillus rhamnosus strain 68.

AIMS: This study focuses on the isolation and characterization of a peptide with bacteriocin-like properties isolated from Lactobacillus rhamnosus strain 68, previously identified by 16S rRNA gene sequencing and originating from human gastrointestinal flora. METHODS AND RESULTS: The peptide was isolated from a supernatant of bacteria maintained under restrictive conditions by a combination of ethanol precipitation and reversed-phase chromatography. The molecular mass of the peptide as assessed by mass spectrometry was 6433.8 Da. An isoelectric point of 9.8 was determined by 2D-PAGE. The peptide designated rhamnosin A inhibited Micrococcus lysodeikticus ATCC 4698 but did not inhibit Lactobacillus plantarum 8014 or Lact. plantarum 39268. Inhibitory activity against M. lysodeikticus at concentrations used in this study was shown to be bacteriostatic rather than bacteriolytic or bactericidal. Rhamnosin A retained biological activity after heat treatment (95 degrees C, 30 min) but was sensitive to proteolytic activity of pepsin and trypsin. CONCLUSIONS: The N-terminal sequence of rhamnosin A, as determined by Edman degradation and in more detail by blast analysis, did not show identity with any currently available Lact. rhamnosus HN001-translated protein sequences, nor any significant similarity with other sequences in the nonredundant protein sequence database. Being a small, heat-stable, nonlanthionine-containing peptide, rhamnosin A should be categorized as a class II bacteriocin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a partial bacteriocin sequence isolated from Lact. rhamnosus 68 and broadens our understanding of bacteriocins.

Purification and characterization of an estrogen-binding peroxidase from human fetuses.

A peroxidase found under two forms with a molecular weight of 220,000 and 170,000 respectively, was purified from human fetuses. The purification procedure included ammonium sulfate precipitation, ion exchange chromatography, gel filtration and hydrophobic interaction chromatography. The purification factor approximated 400. These two forms of peroxidase were found to be immunologically identical as shown when utilizing immunodiffusion. They were able to bind estradiol in the presence of H2O2. This bond resisted to denaturation and solvent extraction therefore suggesting a covalent binding of estradiol to the enzyme.

Human monoclonal IgM DJ binds to ssDNA and human commensal bacteria.

In this study we tried to elucidate further the crossreactivity pattern and binding characteristics of human monoclonal IgM DJ which is an anti-DNA antibody and possesses Y7 natural idiotope. Isolated IgM DJ and its enzymatically obtained fragments Fab' and (Fab')2 were tested for binding to more than 26 antigens and nine bacteria in indirect ELISA. Inhibition of binding studies and examination of the stability of antigen-antibody complexes were also done in ELISA assay. IgM DJ bound to single stranded DNA and human lactic acid bacteria, such as L. acidophyllus, B. bifidum and L. plantarum. This binding was shown to be mediated through IgM DJ Fab' fragment. High avidity and low affinity of interactions was estimated from the binding curves of Fab', (Fab')2 fragments and whole IgM. The common epitopic motif on both antigens were negatively charged phosphodiester moieties. Complexes formed with ssDNA and B. bifidum were resistant to washing with high salt. This suggested that electrostatic attraction was not a strong component of the binding. A novel pattern of natural autoantibody reactivity in a human system related to cross-reactivity with DNA and LAB is described. Possible involvement of LAB in induction of natural anti-DNA antibodies is discussed.

Effect of valency on binding properties of the antihuman IgM monoclonal antibody 202.

A murine monoclonal IgG2a antibody, 202, specific for human IgM, was produced and immunochemically characterized. Binding features of MAb 202, epitope localization, and its accessibility at the quaternary structure of polymeric IgM were investigated. Direct and competitive ELISA with fragments of IgM molecule demonstrated that the epitope recognized by MAb 202 lies on the Fc3 portion of IgM. Sandwich ELISA with MAb 202, which could be used simultaneously to capture and to detect bound IgM, indicated that more than one 202 epitope is present on the IgM molecule. MAb 202 did not precipitate IgM in solution, whereas good precipitation lines were obtained in agarose gel. Binding of MAb 202 to the J chain, C-terminal tailpiece and C mu 2 peptide, which remain attached to the C mu 3 domain of the Fc5 fragment, was excluded by a number of experimental results and structural reasons. Therefore a potential candidate for epitope 202 expression was the C mu 3 domain. MAb 202 did not react with isolated mu chain, which is expected since epitope 202 is of a conformational type. Furthermore, the reaction with monomeric IgM was almost undetectable as was demonstrated by a number of methods (ELISA, immunofluorescence, Western blotting). Since monovalent Fab portions of MAb 202 weakly reacted with polymeric IgM, we concluded that intrinsic affinity of their interaction is low but greatly enhanced by bivalent binding. Antipolymeric IgM binding specificity of MAb 202 was demonstrated only in the case of bivalent binding with a functional affinity constant of Kd = 2.14 x 10(-9) M-1. This implied up to a 10(4) difference between intrinsic and functional affinity, as in the range of concentration used in this study MAb 202 did not react with monomeric IgM.

Impact of magnification views on the characterization of microcalcifications in digital mammography.

PURPOSE: To evaluate the additional benefit of true geometric (air-gap) magnification views for the characterization of microcalcifications in digital mammography. MATERIALS AND METHODS: After ethical approval, we retrospectively reviewed patient records to identify 100 patients with suspicious microcalcifications (35 malignant, 65 benign) who had a standard digital mammography and an additional digital magnification view in the same projection within three months. All images were obtained using an amorphous silicon-based full-field digital system (Senographe 2000 D, GE Healthcare, Chalfont St. Giles, UK). Images were independently analyzed by six board-certified radiologists. The probability of malignancy was estimated using first standard contact mammography alone (MG) and then mammography in combination with the magnification view (MG+MAG) using a modified Breast Imaging Reporting and Data System (BI-RADS) classification system and a percentage scale. Results were compared using receiver operating characteristic (ROC) analysis. In addition, readers assessed the subjective visibility of the calcifications. RESULTS: For all six readers combined, the area under the curve (AUC) was 0.664 ±â€Š0.052 for MG and 0.813 ±â€Š0.042 for MG + MAG, resulting in a statistically significant improvement of 0.148 ±â€Š0.120. Each reader had a higher AUC for MG + MAG than MG, with the improvement being statistically significant in four of the six readers. In 76.34 % of the cases, MG + MAG resulted in better visibility of calcifications compared with mammography alone. In 33 % slightly more and in 39 % significantly more calcifications were found. CONCLUSION: Even in digital mammography with the option of using electronic magnification (zoom) at the viewing workstation, true geometric (air-gap) magnification views remain important for the visibility and correct classification of microcalcifications and for the assessment of their extent.

Diaphragmatic breathing exercises and pelvic floor retraining in children with dysfunctional voiding.

BACKGROUND: Dysfunctional voiding (DV) in neurologically normal children is characterized by involuntary intermittent contractions of either the striated muscle in external urethral sphincter, or the pelvic floor during voiding. Urinary incontinence, pelvic holding maneuvers, voiding difficulties, urinary tract infections (UTIs), constipation and vesicoureteral reflux are highly associated with DV. AIM: To investigate the role of abdominal and pelvic floor muscle (PFM) retraining in children with DV. DESIGN: Prospective clinical controlled study SETTING: Outpatient clinical facility POPULATION: Forty-three children, 5-13 years of age, with dysfunctional voiding METHODS: In addition to standard urotherapy (education, timed voiding, adequate fluid intake, voiding posture and pattern, constipation management and hygiene issues), children were assigned abdominal and PFM retraining. Diaphragmatic breathing exercises were done in lying and sitting positions, for the purpose of achieving abdominal muscle relaxation. PFM retraining consisted of low-level three-second contractions followed by thirty-second relaxation periods. Selected children received pharmacotherapy (anticholinergics or desmopressin). Recurrent symptomatic UTIs were treated with antibiotic prophylaxis. Uroflowmetry with PFM electromyography and ultrasound residual urine volumes were obtained before and at the end of the 12-month treatment period. Clinical manifestations and uroflowmetry parameters were analysed before and after the therapy. RESULTS: After one year of therapy, urinary incontinence was cured in 20 out of 24 patients (83%), nocturnal enuresis in 12 out of 19 children (63%), while 13 out of 19 children (68%) were UTI free. All 15 patients recovered from constipation. Post-treatment uroflowmetry parameters showed significant improvements and a bell-shaped curve was observed in 36 out of 43 children. CONCLUSION: In combination with standard urotherapy, abdominal and pelvic floor muscle retraining is beneficial for curing urinary incontinence, nocturnal enuresis and UTIs in children with DV, as well as for normalizing urinary function. Further trials are needed to define the most effective treatment program which would result in the best treatment outcome. CLINICAL REHABILITATION IMPACT: To improve clinical and objective treatment outcome in dysfunctional voiders. Diaphragmatic breathing and pelvic floor muscle exercises are simple and easy to learn and could be assigned to children aged 5 or older. As they do not require special equipment, they can be performed at all health care levels.

Formes multiples de la lysyl-tARN-ligase de levure et leurs sous-unites.

When Yeast lysyl-tRNA-ligase is extracted in the presence of protease inhibitor (PMSF) a homogeneous species of enzyme is obtained, the subunits of which have a mass of 70 000 daltons. Endogenous degradation produces lighter active species of the enzyme, dimers with subunits in the range 50 000-60 000 daltons. Partial trypsinolysis mimics this degradation, which is therefore thought to occur through proteolysis.

Localization of a protease in protoplast preparations in infected cells of French bean nodules.

Protoplasts from infected and uninfected cells were isolated from the central nitrogen fixing tissue of French bean (Phaseolus vulgaris L. cv Contender) root nodules. Successive filtrations allowed the separation of the infected cells, whereas the small uninfected cells were isolated on a discontinuous Percoll gradient. Higher yields of intact protoplasts were obtained from young (4-week-old) nodules whereas no protoplasts could be isolated from the oldest nodules. When proteolysis was determined in the cytosolic fraction of both infected and uninfected cells, at pH 5.0 and 8.0, with leghemoglobin or azocasein as substrate, activity was present only in infected cell protoplasts and increased with nodule age. A protease with an acidic pH optimum, mainly responsible for this increasing activity, was highly purified from senescing nodules by electro-elution after nondenaturing polyacrylamide gel electrophoresis and used to produce polyclonal antibodies. Western blots of nodule protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with purified anti-protease immunoglobulin G showed the molecular mass of the protease to be 58 kilodaltons. Blots also confirmed that protease protein was located in infected cell protoplasts only, regardless of nodule age.

Possible involvement of nodule superoxide dismutase and catalase in leghemoglobin protection.

Superoxide anion is able to oxidize oxyleghemoglobin prepared from soybean nodules. Furthermore, ferrileghemoglobin is oxidized to leghemoglobin (IV) by hydrogen peroxide and this irreversible reaction leads to a complete inactivation of the hemoprotein. In scavenging O 2 (-) and H2O2, superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) are able to limit these oxidations. The occurrence of these enzymes within soybean nodules and their main characteristics are reported here. A general scheme taking into account their roles in leghemoglobin protection in vivo is proposed.

Detection of cross-reacting idiotypes in sera of lymphoma patients by inverse monoclonal radioimmunoassay.

An anti-idiotypic IgG1 kappa murine monoclonal antibody (MoAb) Y7 against purified monoclonal IgM lambda 1, derived from a patient with Waldenström's macroglobulinemia, has been generated. This antibody cross-reacted with the tumor-derived idiotypes of patients with B cell non Hodgkin's lymphoma as measured by competitive inverse solid radioimmunoassay using unpurified serum samples. Our results with the inhibition curves of 10 sera of normal donors and 60 sera of lymphoma patients indicate that 21 lymphoma patients revealed cross-reactivities greater than 7%, the mean value observed in normal donors. Of these, 5 sera cross-reacted strongly, in the range of 43-163%, revealing a frequency of positive cross-reactivity for MoAb Y7 of 1/12 sera of lymphoma patients. The generation of a panel of anti-idiotypic antibodies which cross-react with different tumor-derived Ig in serum may be valuable for monitoring the disease in a high proportion of NHL patients.

Immunochemical characterisation of a murine monoclonal anti-idiotypic antibody.

Y7, a murine monoclonal IgG1 kappa antibody against a human monoclonal IgM lambda DJ molecule, was affinity purified on an IgM lambda immunoaffinity column. As detected by enzyme-linked immunosorbent assay (ELISA) the isolated Y7 monoclonal antibody was shown to be not cross-reactive with human IgG, human secretory IgA, mu chain, lambda + kappa chains and another human monoclonal IgM lambda BR. Binding to the polyclonal human IgM standard in the same assay was about 30 percent. The epitope specificity of affinity purified and biotinylated Y7 MoAb was localized only in the nonreduced pepsin Fab fragments of IgM lambda DJ immunogen. As the immunogen was determined to be a specific antibody to phosphorylcholine, the specificity of Y7 MoAb was further ascertained in its capacity to induce 95% inhibition of immunogen binding for phosphorylcholine.