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Aims To assess the incremental value of biomarkers, including N-terminal prohormone of brain natriuretic peptide (NT-proBNP), high-sensitivity troponin T (hs-TnT), high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), growth differentiation factor 15 (GDF-15), and procollagen type 1 N-terminal propeptide (P1NP), in predicting incident cardiovascular events and mortality among asymptomatic individuals from the general population, beyond traditional risk factors, including fasting glucose and renal function (cystatin C), medication use, and echocardiographic measures. Methods and results Prospective population-based cohort study of 1324 subjects without a previous cardiovascular event, who underwent baseline echocardiography and biomarker assessment between 2002 and 2006. The clinical endpoint was the composite of myocardial infarction, invasively treated stable/unstable ischemic heart disease, heart failure, stroke, or all-cause mortality. Predictive capabilities were evaluated using Cox proportional-hazards regression, Harrell's concordance index (C-index), and net reclassification improvement. Median age was 66 (interquartile range: 60-70) years, and 413 (31%) were female. During median 8.6 (interquartile range: 8.1-9.2) follow-up years, 368 (28%) composite events occurred. NT-proBNP, hs-TnT, GDF-15, and IL-6 were significantly associated with outcome, independently of traditional risk factors, medications, and echocardiography ( p

By electro-immunoprecipitation methods it was shown that antigen Tp-4 of Treponema pallidum, antigen TR-c of the Reiter treponeme and 'Common Antigen' of Pseudomonas aeruginosa are immunologically cross-reactive. This finding establishes a taxonomic relationship between T. pallidum and a wide range of bacteria. The cross-reactivity explains some false positive reactions in serodiagnostic treponemal tests for syphilis. The cross-reactivity may also play a role in a possible 'normal' immunity against infection with T. pallidum induced by antigens by the normal human bacterial flora.

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are daily adjusted (entrained) by light via the retinohypothalamic tract (RHT). The RHT contains two neurotransmitters, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP), which are believed to mediate the phase-shifting effects of light on the clock. In the present study we have elucidated the role of PACAP in light-induced phase shifting at early night in hamsters and shown that (i) light-induced phase delay of running-wheel activity was significantly attenuated by a specific PAC1 receptor antagonist (PACAP6-38) or by immunoblockade with a specific anti-PACAP antibody injected intracerebroventricularly before light stimulation; (ii) PACAP administered close to the SCN was able to phase-delay the circadian rhythm of running-wheel activity in a similar way to light; (iii) PACAP was present in the hamster RHT, colocalized with melanopsin, a recently identified opsin which has been suggested to be a circadian photopigment. The findings indicate that PACAP is a neurotransmitter of the RHT mediating photic information to the clock, possibly via melanopsin located exclusively on the PACAP-expressing cells of the RHT.

Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the rpoB gene are known to cause rifampin resistance in Escherichia coli and Mycobacterium tuberculosis , and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family Legionellaceae . Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the Legionella pneumophila rpoB gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the rpoB gene (4,104 bp) and an ORF of 384 bp representing part of the rpoC gene was obtained. A 316-bp DNA fragment in the center of the L. pneumophila rpoB gene, corresponding to a previously described site for mutations leading to rifampin resistance in M. tuberculosis , was sequenced from 18 rifampin-resistant Legionella isolates representing four species ( L. bozemanii , L. longbeachae , L. micdadei , and L. pneumophila ), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family Legionellaceae .

To study the structure and function of the Legionella flagellum, we screened a genomic L. micdadei library in Escherichia coli for expression of the flagellin (Fla) subunit. One recombinant clone, JM105 (pHI5588), producing a truncated Fla protein of 40.5 kDa was identified. The plasmid pHI5588 carried a L. micdadei DNA insert of 5 kb, containing ca 95% of the fla gene. The complete DNA sequence of the L. micdadei fla gene was obtained by combining sequence data from pHI5588 with results using a polymerase chain reaction-based system for genome walking (vectorette PCR). The L. micdadei fla gene shared a high degree of homology with other flagellin genes in the amino- and carboxy termini, whereas the central region was found to be nonconserved. The fla sequence will facilitate the cloning of Fla proteins from other Legionella species and the study of flagella in the pathogenesis of Legionnaires' disease.

A 2.7 kb DNA fragment encoding the 60 kDa common antigen (CA) and a 13 kDa protein of Legionella micdadei was sequenced. Two open reading frames of 57677 and 10456 Da were identified, corresponding to the heat shock proteins GroEL and GroES, respectively. Typical −35, −10, and Shine-Dalgarno heat shock expression signals were identified upstream of the L. micdadei groEL gene. Further upstream, a poly-T region, also a feature of the σ32-regulated Escherichia coli groELS heat shock operon, was found. Despite the high degree of homology of the expression signals in E. coli and L. micdadei , Western blot analysis with an L. micdadei specific anti-groEL antibody did not reveal a significant increase in the amount of the GroEL protein during heat shock in L. micdadei or in the recombinant E. coli expressing L. micdadei GroEL.

Circadian rhythms are generated by endogenous clocks in the central brain oscillator, the suprachiasmatic nucleus, and peripheral tissues. The molecular basis for the circadian clock consists of a number of genes and proteins that form transcriptional/translational feedback loops. In the mammalian gonads, clock genes have been reported in the testes, but the expression pattern is developmental rather than circadian. Here we investigated the daily expression of the two core clock genes, Per1 and Per2, in the rat ovary using real-time RT-PCR, in situ hybridization histochemistry, and immunohistochemistry. Both Per1 and Per2 mRNA displayed a statistically significant rhythmic oscillation in the ovary with a period of 24 h in: 1) a group of rats during proestrus and estrus under 12-h light,12-h dark cycles; 2) a second group of rats representing a mixture of all 4 d of the estrous cycle under 12-h light,12-h dark conditions; and 3) a third group of rats representing a mixture of all 4 d of estrous cycle during continuous darkness. Per1 mRNA was low at Zeitgeber time 0–2 and peaked at Zeitgeber time 12–14, whereas Per2 mRNA was delayed by approximately 4 h relative to Per1. By in situ hybridization histochemistry, Per mRNAs were localized to steroidogenic cells in preantral, antral, and preovulatory follicles; corpora lutea; and interstitial glandular tissue. With newly developed antisera, we substantiated the expression of Per1 and Per2 in these cells by single/double immunohistochemistry. Furthermore, we visualized the temporal intracellular movements of PER1 and PER2 proteins. These findings suggest the existence of an ovarian circadian clock, which may play a role both locally and in the hypothalamo-pituitary-ovarian axis.

Circadian rhythms are daily adjusted to the environmental day/night cycle by photic input via the retinohypothalamic tract (RHT). Recent studies indicate that melanopsin, a newly identified opsin-like molecule, is involved in the light responsiveness of retinal ganglion cells (RGCs) constituting the RHT. In the present study, we examined the expression of melanopsin at the mRNA and protein level during a day/night cycle and during prolonged periods of light and darkness in the retina of albino Wistar rats. We observed a diurnal change in melanopsin, with mRNA level being highest at early subjective night and protein level highest at late subjective day. Prolonged exposure to darkness significantly increased melanopsin mRNA level as early as the first day, and the expression continued to increase during 5 d in darkness. The decrease in mRNA level during exposure to constant light was slower. After 48 h of light, the melanopsin mRNA level was significantly reduced, and an almost undetectable level was found after 5 d. The induction of melanopsin by darkness was even more pronounced if darkness was preceded by light suppression for 5 d. By use of immunohistochemistry, we showed that darkness increased the amount of protein in the dendritic processes, resulting in a dense network covering the entire retina. Constant light decreased melanopsin immunostaining time dependently, beginning in the distal dendrites and progressing to the proximal dendrites and the soma. Our observations suggest that the intrinsic light-responsive RGCs adapt their expression of the putative circadian photopigment melanopsin to environmental light and darkness.

PURPOSE. The putative circadian photoreceptor melanopsin is found in rodents in a subpopulation of intrinsic light-sensitive retinal ganglion cells (RGCs) constituting the retinohypothalamic tract (RHT). The study was conducted to determine whether melanopsin is expressed in the human retina and costored with the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP), a marker for the RHT, projecting to the suprachiasmatic nucleus (SCN). Furthermore, whether melanopsin expression is conserved in retinas of blind patients with severe retinal degeneration was investigated. METHODS. In situ hybridization and immunohistochemistry was used to demonstrate melanopsin synthesis in human eyes of 17 donors and two postmortem hypothalami containing the SCN. The coexistence of melanopsin and PACAP in elements of the retinohypothalamic tract was studied by dual-labeling immunocytochemistry. RESULTS. Melanopsin expression was found in a subpopulation of RGCs located in the ganglion cell layer and displaced in the inner nuclear cell layer. Melanopsin-containing cells comprised approximately 0. 8% of all RGCs, with a distinct morphology characterized by two to four dendritic processes constituting a panretinal network. Melanopsin immunoreactivity was primary present at perikaryal boundaries and neuronal processes and to some extent also in the cytoplasm. PACAP and melanopsin were colocalized in the RGCs and PACAP-containing nerve fibers, seemingly innervating the retinorecipient part of the SCN. Melanopsin-expressing RGCs were conserved in retinas of blind patients with severe degeneration of the outer and/or inner layers. CONCLUSIONS. Given the expression of melanopsin in PACAPcontaining RGCs of the human RHT, this photoreceptor is a likely first base in the chain of events leading to photoentrainment of both normal and blind people. (Invest Ophthalmol Vis Sci. 2004;45:4202‐4209) DOI:10.1167/iovs.04-0313

Despite severe degeneration of its eyes, the blind subterranean mole rat, Spalax, is able to adjust circadian rhythms to the environmental light/dark cycle due to a conserved retinohypothalamic tract (RHT). The photopigment mediating the circadian photoreception and it cellular localisation is unknown in the Spalax retina. Here we show, using in situ hybridization and immunohistochemistry, that melanopsin, a recently identified opsin, is expressed in retinal ganglion cells which also co-store PACAP, a neurotransmitter of the RHT. The melanopsin-component of retinal ganglion cells in the Spalax retina is well conserved resulting in a relatively higher density of melanopsin positive cells per area compared to the rat. The results show that the Spalax, as sighted animals expresses melanopsin in ganglion cells projecting to the circadian clock supporting a role of melanopsin as a circadian photopigment.

Objective and design: This cross-sectional study aimed to investigate associations between a marker of cardiac strain, the N-terminal prohormone B-type natriuretic peptide (NT-proBNP), and inflammation as reflected by either a conventional or novel inflammatory marker in a bi-ethnic South African cohort. Methods and subjects: We measured NT-proBNP, C-reactive protein (CRP) and plasma-soluble urokinase plasminogen activator receptor (suPAR) levels along with conventional biomarkers in black (n = 117) and white (n = 116) men. Results: NT-proBNP, CRP and suPAR levels were higher in black compared to white men. NT-proBNP was significantly associated with both CRP (r = 0.38; p = 0.001) and suPAR (r = 0.42; p,0.001) in black men only. After full adjustment in multiple regression analyses, the above associations of NT-proBNP with CRP (b = 0.199; p = 0.018) and suPAR (b = 0.257; p,0.01) were confirmed in black men. Conclusion: These results suggest that a low-grade inflammatory state as reflected by both a conventional and novel marker of inflammation may contribute to higher cardiovascular risk as reflected by the associations obtained with a marker of cardiac strain in black South African men South African National Research Foundation Grant (GUN 2073040), the Medical Research Council (South Africa) and the Africa Unit for Trans-disciplinary Health Research of the North-West University (Potchefstroom campus, South Africa) http://www.plosone.org http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058506 DOI: 10.1371/journal.pone.0058506

Combining the electrophoretic principles of counter current immunoelectrophoresis and deflection of line precipitates in line immunoelectrophoresis provides a new technique for quantitative determination of antibodies against specific antigens (CCLD electrophoresis), even if no purified antigen or monospecific antibodies are available for construction of the detection system. We have used the method for quantitative determination of antibodies against the flagellum of Treponema phagedenis biotype Reiter and compared the diagnostic potential of this method in the diagnosis of syphilis with an ELISA method for the quantification of IgG antibodies against the flagellum. The CCLD electrophoresis could be optimized to a diagnostic performance very similar to that achieved using the ELISA method.

As the first step in a cooperative effort to standardize the identification of the polypeptides of Treponema pallidum subsp. pallidum, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results obtained in 16 laboratories were compared. Although it was possible to correlate the positions of 16 of the major polypeptide bands, the cross-identification of many of the polypeptides was ambiguous, particularly in the low molecular weight range. Two-dimensional electrophoresis provided an improved means of separating and characterizing T. pallidum polypeptides as isolated molecular species. An approach to the unambiguous identification of treponemal polypeptides was outlined which will utilize two-dimensional electrophoresis in combination with specific properties attributable to individual proteins, including reactivity with monoclonal antibodies or monospecific antisera, biochemical and structural properties, and sequence information. To demonstrate the feasibility of this approach, two-dimensional electrophoresis in conjunction with immunoperoxidase staining was used to specifically identify three cloned T. pallidum proteins.

In gene libraries of Mycobacterium bovis BCG, Mycobacterium tuberculosis and Mycobacterium leprae recombinants were frequently found expressing an immunodominant 65 kDa protein antigen. In this study polyclonal and monoclonal antibodies against the 65 kDa antigen were found to react with a variety of different bacteria. Furthermore it is shown that the 65 kDa mycobacterial protein belongs to the family of antigens previously designated ‘common antigen’ due to their presence in a large variety of bacterial species. The molecular weight of this common antigen in 17 bacterial species was determined and it varied from 59 to 65 kDa. These bacteria included Gram-negative, Gram-positive and archae-bacteria, indicating that this antigen consists of members of an evolutionary well-conserved protein family. The 65 kDa protein was located in the cytoplasmic fraction of both Escherichia coli K12 and M. bovis BCG. Its function for the bacterial cell is presently unknown. The immunological relatedness of this common antigen to the MbaA protein might indicate a role in the etiology of rheumatoid arthritis, as was recently suggested for the mycobacterial 65 kDa antigen.

To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15–25 kilobase pairs (kb)) were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli -absorbed anti-L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly with a monospecific rabbit antiserum raised against L. micdadei“common antigen” (CA), and an additional 13 K L. micdadei protein. The region encoding these two proteins from the 17 kb recombinant plasmid (pBA 2) was subcloned in pBGS 18+. The DNA sequence of the CA encoding region in the 2.7 kb subcloned fragment will provide important information with respect to genetic vs. antigenic relatedness among Legionellae and other Gram-negative species, as well as to CA structure and possible function.

The immunochemical properties of the Treponema pallidum antigen TpD, as expressed by Escherichia coli K12, was investigated by crossed immunoelectrophoresis in which an affinity-purified antibody to this antigen was used. Two immunologically cross-reacting components of TpD with different mobility were demonstrated. Affinity-purified antibodies were used in obtaining purified TpD and in determining the cellular localization of TpD in T. pallidum by immunoelectron microscopy. TpD was localized on the surface of methanol-fixed T. pallidum. Twenty sera from patients with secondary syphilis and 20 sera from nonsyphilitics were examined in crossed immunoelectrophoresis. All sera from patients with secondary syphilis and none from nonsyphilitics contained antibodies to the TpD components. Because TpD seems to be surface associated and a major immunogen during infection with T. pallidum, this antigen might be useful for development of a vaccine against syphilis and for development of improved methods for serodiagnosis of syphilis.

SUMMARY: A library of Treponema pallidum DNA was constructed using a cosmid cloning system. Sixteen hundred Escherichia coli recombinant clones were generated covering the T. pallidum genome with a probability of 99%. Three hundred of the clones were screened for expression of T. pallidum antigens by a modified rocket immunoelectrophoresis technique using a polyspecific antiserum to T. pallidum. One clone was identified which produced the ‘common antigen' (CA) of T. pallidum (Tp-4). CA shares epitopes with antigens present in more than 50 different bacterial species, but nothing is known about its structure, function and localization. The recombinant E. coli clone will be of value for a structural analysis of the CA gene.

Thirteen rabbits were immunized three times at weekly intervals with 50 micrograms of flagella purified from Treponema phagedenis biotype Reiter. Fourteen rabbits were inoculated in the same way with a placebo preparation. Rabbits immunized with the flagella developed an immune response to the flagella but showed no statistically significant prolongation of incubation time or diminution of lesion severity when challenged intradermally with 4 X 10(3) Treponema pallidum organisms.

Quantitative crossed immunoelectrophoresis was used to evaluate the antigenic similarity of Pseudomonas aeruginosa and Pseudomonas cepacia GroEL proteins. We found that the two proteins showed 75% identity. By using a panel of monoclonal antibodies against the P. aeruginosa GroEL protein, we identified 10 monoclonal antibodies which cross-reacted with the P. cepacia GroEL protein and 21 monoclonal antibodies which recognized type-specific epitopes on the P. aeruginosa GroEL protein. In crossed immunoelectrophoresis two different fractions of GroEL reactive material could be resolved. These fractions showed a reaction of partial identity. Examination of the two immunoprecipitates by Western blotting, showed that both fractions consisted of anti-60 kDa GroEL reactive protein. One fraction, in addition, contained LPS with a characteristic ‘ladder’ reaction in modified Western blotting. We therefore conclude that this fraction represents a complex between LPS and GroEL.