Risk of bacteremia in patients presenting with shaking chills and vomiting - a prospective cohort study.

Chills and vomiting have traditionally been associated with severe bacterial infections and bacteremia. However, few modern studies have in a prospective way evaluated the association of these signs with bacteremia, which is the aim of this prospective, multicenter study. Patients presenting to the emergency department with at least one affected vital sign (increased respiratory rate, increased heart rate, altered mental status, decreased blood pressure or decreased oxygen saturation) were included. A total of 479 patients were prospectively enrolled. Blood cultures were obtained from 197 patients. Of the 32 patients with a positive blood culture 11 patients (34%) had experienced shaking chills compared with 23 (14%) of the 165 patients with a negative blood culture, P = 0.009. A logistic regression was fitted to show the estimated odds ratio (OR) for a positive blood culture according to shaking chills. In a univariate model shaking chills had an OR of 3.23 (95% CI 1.35-7.52) and in a multivariate model the OR was 5.9 (95% CI 2.05-17.17) for those without prior antibiotics adjusted for age, sex, and prior antibiotics. The presence of vomiting was also addressed, but neither a univariate nor a multivariate logistic regression showed any association between vomiting and bacteremia. In conclusion, among patients at the emergency department with at least one affected vital sign, shaking chills but not vomiting were associated with bacteremia.

Search for charged Higgs bosons in e+e- collisions at [Formula: see text].

A search is made for charged Higgs bosons predicted by Two-Higgs-Doublet extensions of the Standard Model (2HDM) using electron-positron collision data collected by the OPAL experiment at [Formula: see text], corresponding to an integrated luminosity of approximately 600 pb-1. Charged Higgs bosons are assumed to be pair-produced and to decay into [Formula: see text], τντ or AW±. No signal is observed. Model-independent limits on the charged Higgs-boson production cross section are derived by combining these results with previous searches at lower energies. Under the assumption [Formula: see text], motivated by general 2HDM type II models, excluded areas on the [Formula: see text] plane are presented and charged Higgs bosons are excluded up to a mass of 76.3 GeV at 95 % confidence level, independent of the branching ratio BR(H±â†’τντ ). A scan of the 2HDM type I model parameter space is performed and limits on the Higgs-boson masses [Formula: see text] and mA are presented for different choices of tanß.

Induction of anti-ß2 -glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes.

BACKGROUND: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti-ß(2) -glycoprotein I (ß(2) -GPI) autoantibodies. ß(2) -GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish-hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. ß(2) -GPI has been shown to interact with Streptococcus pyogenes. OBJECTIVE: To evaluate the potential of S. pyogenes-derived proteins to induce anti-ß(2) -GPI autoantibodies. METHODS AND RESULTS: Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen-like protein A [SclA], and streptococcal collagen-like protein B [SclB]) were found to interact with ß(2) -GPI. Only binding to protein H induces a conformational change in ß(2) -GPI, thereby exposing a cryptic epitope for APS-related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody-related epitope in domain I of ß(2) -GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti-protein H antibodies also generated anti-ß(2) -GPI antibodies. CONCLUSIONS: Our study has demonstrated that a bacterial protein can induce a conformational change in ß(2) -GPI, resulting in the formation of antiß(2) -GPI autoantibodies. This constitutes a novel mechanism for the formation of anti-ß(2) -GPI autoantibodies.

Antibodies against four proteins from a Streptococcus pyogenes serotype M1 strain and levels of circulating mannan-binding lectin in acute poststreptococcal glomerulonephritis.

BACKGROUND: Responses against antigens from the potentially nephritogenic Streptococcus pyogenes serotype M1 in patients with acute poststreptococcal glomerulonephritis (AGN) were studied to seek indications of expression of these antigens during the preceding infection. Also, the question was asked whether the complement protein mannan-binding lectin (MBL) is required for development of the hypocomplementemia associated with AGN. Hypothetically, the lectin pathway might trigger the alternative pathway, which is consistently activated in AGN. METHODS: Antibodies against three proteins associated with M1, M1 protein, streptococcal inhibitor of complement (SIC) and protein H, an IgG-binding protein, were determined by ELISA in 56 children and 17 adults with AGN. Antibodies against streptococcal cysteine proteinase, which is produced by all serotypes of S. pyogenes, were also examined. MBL concentrations were measured in the same 71 patients by a sandwich ELISA. RESULTS: Increased concentrations of antibodies were found against all four streptococcal proteins, albeit not uniformly distributed between different subgroups of patients. The prevalence of low MBL concentrations (<100 microg/l) including 2 patients with undetectable MBL (<10 microg/l) was similar in AGN (11%) and in controls (16%). CONCLUSIONS: Our results give evidence of exposure to SIC and protein H in conjunction with AGN. This implies that SIC and protein H and/or cross-reacting proteins may have a role in the pathogenesis of AGN or that streptococci expressing SIC or protein H are nephritogenic for other reasons. The finding of MBL-deficient individuals among the patients demonstrates that MBL is not necessary for the recruitment of complement in AGN.

Behavioral and neurochemical consequences of repeated nicotine treatment in the serotonin-depleted rat.

RATIONALE: Repeated exposure to addictive drugs causes neuroadaptive alterations that are proposed to increase the incentive motivation to consume drugs and to decrease the ability to inhibit such inappropriate motivational impulses and responses. Together, these behavioral consequences of drug intake may underlie the compulsive drug-seeking and -taking behaviors observed in drug abuse. OBJECTIVE: Brain serotonin (5-HT) has been implicated in these mechanisms and this study therefore investigated the consequences of brain 5-HT depletion on the behavioral and neurochemical effects induced by repeated daily nicotine treatment (15 days) in male rats. METHODS: The effects of the present pharmacological manipulations were evaluated behaviorally (locomotor activity, the elevated plus-maze) and neurochemically (microdialysis, brain biochemistry). RESULTS: Depletion of brain 5-HT produced behavioral disinhibition in the elevated plus-maze. In 5-HT-depleted animals, nicotine-induced locomotor sensitization was observed on treatment days 5, 10, and 15, but only on day 15 in the sham-operated rats. Postsensitization, the locomotor stimulatory effects of amphetamine and the dopamine receptor agonists SKF 38,393, apomorphine, and quinpirole were decreased in 5-HT-depleted animals, an effect that appeared to be more pronounced in nicotine-treated rats. Repeated nicotine treatment sensitized the nicotine-induced elevation of the extracellular accumbal dopamine levels in sham-operated, but not in 5-HT-depleted rats, and was also associated with decreased D2 autoreceptor function in both nicotine-treated experimental groups. CONCLUSIONS: Depletion of brain 5-HT, which produces behavioral disinhibition, may slightly facilitate the overall expression of locomotor sensitization to nicotine and differentially affect the pre- and postsynaptic neuroadaptive events involved in the expression of these phenomena.

Effects of 5-HT1A and 5-HT2 receptor agonists on the behavioral and neurochemical consequences of repeated nicotine treatment.

This study investigated the effects of repeated daily (15 days) treatment with nicotine, alone or in combination with the 5-HT1A/7 receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) or the 5-HT2 receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI) on locomotor sensitization, mesolimbic dopamine neurochemistry and on behavioral inhibition in the rat. Acute nicotine elevated the extracellular dopamine levels in the nucleus accumbens and stimulated locomotor activity, effects that were sensitized after repeated nicotine treatment. Repeated nicotine administration also produced nicotine-induced behavioral disinhibition in the elevated plus-maze. Treatment with DOI counteracted the expression of the nicotine-induced locomotor and neurochemical sensitization, but had no effect on nicotine-induced behavioral disinhibition. Treatment with 8-OH-DPAT decreased the expression of nicotine-induced behavioral disinhibition, but had no effect on locomotor or neurochemical sensitization. Taken together, these findings suggest that the 5-HT1A and the 5-HT2 receptor subtypes are differentially involved in the effects of repeated nicotine on locomotor sensitization, behavioral inhibition and mesolimbic dopamine neurochemistry.

Systemic vasculitis in a kidney transplant population.

BACKGROUND: Systemic vasculitis as original disease might adversely influence the result of kidney transplantation. METHODS: The clinical course after 32 transplantations to 26 patients with microscopic polyangiitis, Wegener's granulomatosis, Henoch-Schonlein purpura, thrombotic thrombocytopenic purpura/hemolytic uremic syndrome, or Goodpasture's disease was evaluated. The median follow-up time was 82 months (range, 4-132 months). Frozen sera from 25 transplantations were analyzed for Goodpasture antibodies, myeloperoxidase antineutrophil cytoplasmic antibodies (ANCA), and proteinase 3 ANCA. RESULTS: Survival of patients and grafts did not differ between patients and matched controls. Recurrent vasculitis occurred with seven grafts (four patients with microscopic polyangiitis or Wegener's granulomatosis, two patients with Henoch-Schonlein purpura, and one patient thrombotic thrombocytopenic purpura). New-onset hematuria was the initial renal symptom in five patients. Treatment with corticosteroids, cyclophosphamide, and/or plasma exchange was most often effective, but two grafts were lost. Proteinase 3 ANCA titers were increased to 12-738 U/ml before seven transplants. The patient with the lowest titer lost his graft due to recurrence, two other patients had reversible recurrence after 1 year and 5 years, two patients lost their grafts due to unknown/unrelated causes, and two patients' grafts remain without recurrence. Myeloperoxidase ANCA were increased to 22-39 U/ml before two transplants, which have been uneventful for 4 years. CONCLUSIONS: An awareness of the small but perpetual risk of recurrence facilitates early treatment that may save the transplant. Testing for hematuria and early transplant biopsies, and possibly monitoring of ANCA titers, are essential, but pretransplant ANCA titers have no predictive value in asymptomatic patients. Results of kidney transplantation in patients with vasculitis are as good as in other patients.

Protein SIC, a novel extracellular protein of Streptococcus pyogenes interfering with complement function.

The human pathogen Streptococcus pyogenes possesses a chromosomal region, the mga regulon, that contains co-regulated genes important to the virulence of these bacteria. A novel gene located in the mga regulon of a S. pyogenes strain of serotype M1 was cloned and sequenced. It translates into a protein of 305 amino acid residues, including a signal sequence of 32 amino acids and a central region consisting of three tandem repeats. The sequence represents a novel structure with no significant homology to any previously published sequence. The protein was purified from the streptococcal culture media where it is present in substantial amounts. Affinity chromatography of human plasma on Sepharose coupled with the protein specifically absorbed two plasma proteins which were identified as clusterin and histidine-rich glycoprotein (HRG). The interactions between the streptococcal protein and the plasma proteins were further characterized using purified clusterin and HRG. Inhibition experiments indicated that they have affinity for overlapping or closely located sites in the streptococcal protein. Both clusterin and HRG are regulators of the membrane attack complex (C5b-C9) of complement. When the streptococcal protein was added to serum, complement-mediated lysis of sensitized sheep erythrocytes and guinea pig erythrocytes was inhibited. In addition, the streptococcal protein was incorporated into C5b-C9 in serum, indicating the location of its action. The name, protein SIC, streptococcal inhibitor of complement-mediated lysis, is therefore suggested for this novel protein. The occurrence of protein SIC and its gene was investigated in a collection of S. pyogenes strains comprising 55 different M serotypes. Only M1 and M57 strains were positive in this screening, indicating that protein SIC could be a virulence determinant. Thus, during recent years, the M1 serotype has been connected with a world-wide increase of severe and toxic S. pyogenes infections.

Allosteric and temperature effects on the plasma protein binding by streptococcal M protein family members.

Most group A streptococcal strains bind immunoglobulins (Ig) and fibrinogen to their cell walls. It is shown in this paper that the Ig-binding of three different strains was much weaker at 37 degrees C than at room temperature (20 degrees C), whereas the fibrinogen binding was unaffected by temperature. The binding properties and molecular sizes of two purified group A streptococcal cell surface proteins from the M protein family were studied at various temperatures, M1 protein with affinity for IgG, fibrinogen and albumin, and protein Sir22 with affinity for IgA and IgG. Both proteins appeared as monomers which bound all their ligands, including fibrinogen, very weakly at 37 degrees C, and as strongly binding dimers at 10 and 20 degrees C. Furthermore, the results demonstrated that the plasma protein binding of the bacterial proteins was allosterically regulated, i.e. the binding of a ligand to one site modulated the binding of a ligand to a second site. For example, the binding of albumin or IgG to purified M1 protein at 10 and 20 degrees C strongly enhanced the binding of fibrinogen at 37 degrees C. This indicates that the high affinity dimer form of the bacterial proteins can be stabilized at 37 degrees C, a possible explanation for the strong fibrinogen binding of whole bacteria. Finally, the sizes and binding properties of three M1 protein fragments were studied and the results indicated that the centrally located C-repeats, which are conserved among the members of the M protein family, are important for the formation of the high-affinity dimers of the bacterial proteins.

Structure and stability of protein H and the M1 protein from Streptococcus pyogenes. Implications for other surface proteins of gram-positive bacteria.

M proteins and other members of the M protein family, expressed on the surface of Streptococcus pyogenes, bind host proteins such as immunoglobulins, albumin, and fibrinogen. Protein H and the M1 protein are expressed by adjacent genes and both belong to the M protein family. In this work, the structure and stability of these two proteins have been investigated. As judged from sequence analysis and circular dichroism spectroscopy, the proteins are almost entirely in an alpha-helix conformation. The amino acids are arranged in a seven-residue (heptad) repeat pattern along the greater part of the proteins. These observations support the previously accepted model of M proteins as coiled-coil dimers. However, it was also found that the structures of both proteins were thermally unstable; i.e., the content of helix conformation was greatly reduced at 37 degrees C as compared to 25 degrees C or below. Together with previous findings that these proteins appear as monomers at 37 degrees C and dimers at low temperatures, the results suggest that the coiled-coil dimers are unfolded at 37 degrees C. The heptad patterns of protein H and the M1 protein showed a nonoptimal distribution of residues expected for a coiled-coil conformation. This is a possible explanation for the low thermal stability of the proteins. It was also demonstrated that the proteins were stabilized in the presence of the ligands IgG and/or albumin. Protein H and M1 protein show a high degree of sequence similarity in their C-terminal regions, and a fragment from this region displayed a high content of helix conformation, whereas fragments from the nonsimilar N-terminal parts did not adopt any stable folded structure. Thus, the C-terminal parts, which are conserved within the M protein family, may constitute a framework for the formation of the parallel helical coiled-coil structure, and we propose that the less stable N-terminal part may also participate in antiparallel interaction with M proteins on adjacent bacteria. The results suggest that temperature fluctuations in the environment could change the properties of bacterial surface proteins, thereby affecting the molecular interactions between the bacterium and its host.

M1 protein and protein H: IgGFc- and albumin-binding streptococcal surface proteins encoded by adjacent genes.

M1 protein and Protein H are surface proteins simultaneously present at the surface of certain strains of Streptococcus pyogenes, important pathogenic bacteria in humans. The present study concerns the structure, protein-binding properties and relationship between these two molecules. The gene encoding M1 protein (emm1) was found immediately upstream of the Protein H gene (sph). Both genes were preceded by a promoter region. Comparison of the sequences revealed a high degree of similarity in the signal peptides, the C repeats located in the central parts of the molecules and in the C-terminal cell-wall-attached regions, whereas the N-terminal sequences showed no significant similarity. Protein H has affinity for the Fc region of IgG antibodies. Also M1 protein, isolated from streptococcal culture supernatants or from Escherichia coli expressing emm1, was found to bind human IgGFc. When tested against polyclonal IgG from eight other mammalian species, M1 protein and Protein H both showed affinity for baboon, rabbit and pig IgG. M1 protein also reacted with guinea-pig IgG, whereas both streptococcal proteins were negative in binding experiments with rat, mouse, bovine and horse IgG. The two proteins were also tested against other members of the immunoglobulin super family: human IgM, IgA, IgD, IgE, beta 2-microglobulin, and major histocompatibility complex (MHC) class-I and class-II antigens. M1 protein showed no affinity for any of these molecules whereas Protein H reacted with MHC class-II antigens. M1 protein is known to bind albumin and fibrinogen also. The binding sites for these two plasma proteins and for IgGFc were mapped to different sites on M1 protein. Thus albumin bound to the C repeats and IgGFc to a region (S) immediately N-terminal of the C repeats. Finally, fibrinogen bound further towards the N-terminus but close to the IgGFc-binding site. On the fibrinogen molecule, fragment D was found to mediate binding to M1 protein. The IgGFc-binding region of M1 protein showed no similarity to that of Protein H. Still, competitive binding experiments demonstrated that the two streptococcal proteins bound to overlapping sites on IgGFc.

Protein H--a surface protein of Streptococcus pyogenes with separate binding sites for IgG and albumin.

Protein H, a molecule expressed at the surface of some strains of Streptococcus pyogenes, has affinity for the constant (IgGFc) region of immunoglobulin (Ig) G. In absorption experiments with human plasma, protein H-sepharose could absorb not only IgG but also albumin from plasma. The affinity constant for the reaction between albumin and protein H was 7.8 x 10(9) M-1, which is higher than the affinity between IgG and protein H (Ka = 1.6 x 10(9) M-1). Fragments of protein H were generated with deletion plasmids and polymerase chain reaction (PCR) technology. Using these fragments in various protein-protein interaction assays, the binding of albumin was mapped to three repeats (C1-C3) in the C-terminal half of protein H. On the albumin molecule, the binding site for protein H was found to overlap the site for protein G, another albumin- and IgGFc-binding bacterial surface protein. Also IgGFc-binding could be mapped with the protein H fragments and the region was found N-terminally of the C repeats. A synthetic peptide (25 amino acid residues long) based on a sequence in this region was shown to inhibit the binding of protein H to immobilized IgG or IgGFc. This sequence was not found in previously described IgGFc-binding proteins. However, two other cell surface proteins of S. pyogenes exhibited highly homologous regions. The results identify IgGFc- and albumin-binding regions of protein H and further define and emphasize the convergent evolution among bacterial surface proteins interacting with human plasma proteins.

Antibody response in immunized rabbits measured with bacterial immunoglobulin-binding proteins.

Protein G, an immunoglobulin (Ig)-binding protein isolated from group C or G streptococci, binds to the Fc portion of IgG. Protein L, from the anaerobic bacterium Peptostreptococcus magnus, specifically binds light chains of Ig. In this study, protein G and L were used to measure the production of antibodies in immunized rabbits. Two rabbits were immunized with a mixture of human urinary proteins from a patient with tubular proteinuria, and blood samples were collected regularly from the animals for 6 weeks after the immunization. The antibody levels of the blood samples against six of the proteins in the antigen mixture were then measured by ELISA. Microtiter plates were coated with each of the antigens, incubated with the rabbit serum samples, and the specific antibodies of the IgG class measured by incubation with biotinylated protein G, and antibodies of all Ig classes with biotinylated protein L. Alternatively, Western blotting was employed, where the antibodies which bound to each antigen after separation by SDS-PAGE and transfer to nitrocellulose membranes, were detected by protein G or L. The results showed that antibody production against five of the antigens, albumin, alpha 1 gamma-acid glycoprotein, alpha 1 gamma-microglobulin, Ig light chains, and retinol-binding protein, showed a similar pattern, although the magnitude of the initial IgM response differed somewhat. After 6 weeks, the levels of the protein G-binding antibodies had reached a plateau, while those of protein L-binding antibodies were still increasing. The response to the sixth antigen, beta 2 microglobulin, was considerably different. A dramatic increase of anti-beta 2 gamma-microglobulin antibodies was seen during the 4th week after immunization when protein L was used.

Protein H--a novel IgG binding bacterial protein.

Forty-eight group A streptococcal strains of different M types were screened for binding of human radiolabeled IgG. Three of the strains bound more than 80% of the added radioactivity and one of them, an M protein type 1 strain designated AP1, was selected for further analysis. Attempts were made to solubilize the IgG binding bacterial molecule, and small amounts of an IgG binding protein with a mol. wt of 40 kDa could be solubilized with mutanolysin, a muramolytic agent. The gene encoding this streptococcal protein was cloned and expressed in E. coli, and the E. coli-produced protein was purified in a single step by affinity chromatography on IgG-Sepharose. When tested with IgGs from different species, the molecule was found to bind human IgG almost exclusively. The N-terminal amino acid sequence was determined and showed no homology with previously isolated Ig binding proteins, and the name protein H (as in human IgG) is suggested for this novel Ig binding bacterial protein. Protein H showed preferential affinity for heavy chains and Fc fragments of human IgG, and did not bind Ig light chains. The affinity constant, determined by Scatchard plots, between protein H and human polyclonal IgG was 1.6 x 10(9). No binding was observed between protein H and IgM, IgA, IgD, or IgE. Finally, when tested against several additional proteins and human plasma, protein H only showed weak binding to alpha 2-macroglobulin, a proteinase inhibitor.

Bacterial growth blocked by a synthetic peptide based on the structure of a human proteinase inhibitor.

Cysteine proteinases are important not only in the intracellular catabolism of peptides and proteins and in the processing of prohormones and proenzymes, but also in the penetration of normal human tissue by malignant cells and possibly microorganisms, including viruses. Cystatin C is a human cysteine proteinase inhibitor present in extracellular fluids. We have synthesized peptide derivatives mimicking the proposed proteinase-binding centre of cystatin C and find that they irreversibly inhibit cysteine proteinases. Several bacteria produce proteinases, so we tested a tripeptide derivative (Z-LVG-CHN2) for in vitro anti-bacterial activity against a large number of bacterial strains belonging to thirteen different species. It was found to inhibit specifically the growth of all strains of group A streptococci. The susceptibility of these human pathogens to the peptide was compared with that to well-established anti-streptococcal antibiotics such as tetracycline and bacitracin. Moreover, the peptide was active in vivo against group A streptococci: mice injected with lethal doses of these bacteria were cured by a single injection of Z-LVG-CHN2. The cysteine proteinase produced by group A streptococci was isolated and found to be inhibited by Z-LVG-CHN2; moreover, excess proteinase relieved the growth inhibition caused by the peptide derivative, suggesting that the antibacterial activity of Z-LVG-CHN2 is due to inhibition of this cysteine proteinase. This strategy of blocking proteinases with peptide derivatives that mimic naturally occurring inhibitors could be useful in the construction of new agents against other microorganisms, including viruses.