Preparation method of lamprey antisera and activity assay.

Lampreys, the surviving representative of jawless vertebrates, have been a focal point in the search for the evolutionary origin of adaptive immunity. They have independently evolved the variable lymphocyte receptor (VLR)-based adaptive immune system that protects themselves from infection by a variable of microorganisms. The standard immunization schedule for Japanese lamprey (Lampetra japonica) was established to prepare antisera by injection of Escherichia coli, Bacillus proteus, Staphylococcus aureus, Mycobacterium smegmatis, RRBCs, SRBCs, NB4 cells and Hela cells. In this study, we demonstrated the activities of lamprey antisera, which might be helpful to research the collaboration between VLR-based adaptive immune system and complement system in jawless vertebrates.

The prevailing O serogroups among the serologically differentiated clinical Proteus spp. strains in central Poland.

In the years 2006-2011, 617 Proteus spp. strains isolated mostly from urine and wounds or other clinical sources were collected in Lódz, Poland, to determine the offensive O serotypes frequently occurring among patients. P. mirabilis exhibited the most intensive swarming growth and was dominating species (86.9%), followed by P. genomospecies, P. vulgaris, and P. penneri. Ninety four per cent strains were recognized as S (smooth) forms. Serological studies (involving ELISA-enzyme-linked immunosorbent assay and Western blotting using native and adsorbed rabbit antisera) enabled classification of 80% S isolates into respective Proteus O serogroups among the 83 ones, described so far. The remaining strains seemed to be serologically unique. Despite the observed big serological variety of Proteus spp. isolates, we found the O78 serogroup recently described in Poland as dominating and identified other widespread serotypes: O3, O6, O10, O11, O27, O28, and O30 reported earlier as predominating also in other countries; O77 and O79 detected lately in Poland; O16, O18, O20, and O50. No unique structural feature of the prevalent O serotypes has been indicated. However, the prevalence of some O serogroups indicates that particular serotypes may be in some ways beneficial to the strains producing these kinds of O antigen.

Antibodies to photoproducts of deoxyribonucleic acids irradiated with ultraviolet light.

A rabbit immunized with complexes of methylated bovine serum albumin and ultraviolet-irradiated DNA from calf thymus produced antibodies directed toward the photoproducts in the DNA. Serologic activity appeared after irradiation of DNA at 270 mmicro and decreased upon irradiation at 235 mmicro. The antigenic determinants of the ultraviolet-treated DNA appear to be photoproducts associated primarily with thymine, as measured by direct dependence of serologic activity on the adenine-thymine content of the DNA, and by inhibition of the Serlolgic reaction by the irradiated di-,tri-,and tetra-(thymidine-5'-phosphate nucleotides.

Cross-reaction between proteins of Larrea divaricata Cav. (jarilla) and proteins of Gram-negative bacteria.

Larrea divaricata is an abundant plant of northwest of Argentina used to treat different pathologies. We aimed to characterize the immunogenicity of proteins from a partially purified crude aqueous extract (JPCE) of jarilla. We evaluated the cross reaction between JPCE and whole cell-bacterial proteins (W-CBP) of Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Klebsiella pneumoniae using a mouse anti-JPCE serum. Protein profiles of JPCE and W-CBP were analyzed. For JPCE, 18 bands were observed in a 20-176 kDa range. Levels of IgG against JPCE and W-CBP were determined. Bacterial proteins showed a strong reaction with the anti-JPCE serum. Plant proteins could be used as immune stimulants.

The cross-reactivity of Shewanella fidelis lipopolysaccharide with anti-Proteus antibodies.

The serological cross-reactivity between lipopolysaccharides (LPS) of S. fidelis KMM3582(T) and rabbit anti-O P. mirabilis antibodies was tested. Using ELISA and Western blot cross-reactivity between S. fidelis LPS and antisera against P. mirabilis O14, O3 LPSs was found. The observed cross-reaction may suggest that anti-P. mirabilis S1959 (O3) antibodies may bind to the internal part of S. fidelis O-polysaccharides. A weak interaction between S. fidelis LPS and antiserum against P. mirabilis O13 in Western blot suggests that the absolute configuration of non-sugar "AlaLys" component (N(epsilon)-[(S)-l-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine) may influence the affinity of antibodies for S. fidelis LPS.

Anti-tumor preventative effect of mono-therapy with the use of proteus vaccine, Staphylococcus antitoxin and divaccine of Staphylococcus-proteus.

Anti-tumor preventative effect of mono-therapy with the use of Proteus vaccine, Staphylococcus antitoxin and divaccine of Staphylococcus-Proteus has been studied. Experiments were carried out on 80 non-purebred laboratory white mice (age--3-3,5 months, body mass--18-20 g) and 60 rats (body mass--100-120 g) using intraperitoneal inoculation of Ehrlich's adenocarcinoma (ascitic form--EAT, in mice, cancer cells--3 x 10(6)), and subcutaneous inoculation of Sarcoma S-45 (in rats). Anti-tumor preventative effect of bacterial vaccines and immunization was evaluated according to the following parameters: Frequency of cancer development, Inhibition of cancer growth, Body mass index of experimental animals, Volume of ascitic fluid. Results of experiments have shown that use of bacterial polysaccharides with preventative purposes has better effect at S-45 growth than at EAT growth; Vaccination with Proteus prolongs lifespan mach more than vaccination with antitoxin of Staphylococcus; Vaccination with complex divaccine of Staphylococcus-Proteus causes complete resorption of tumors from 32 to 60 days; Development of experimental malignant tumors depends on type of anti-microbial vaccines and starting date of inoculation after completion of vaccination.

Microfluidic chip analysis of outer membrane proteins responsible for serological cross-reaction between three Gram-negative bacteria: Proteus morganii O34, Escherichia coli O111 and Salmonella Adelaide O35.

Bacterial strains have complex and individual antigenic structure, which provides basis for their serological identification. However, serological cross-reaction may occur when antibodies against a certain strain recognize other strains too. The molecular basis of this phenomenon is the expression of similar or identical antigenic epitopes on the surface of different bacterial cells. Such cross-reactions might harden the serological diagnosis of pathogenic bacteria. But it can be also advantageous, when antigens of non-pathogenic strains can be used in the serological examinations. Serological cross-reaction between three taxonomically different strains--Proteus morganii O34 (8662/64), Escherichia coli O111 and Salmonella Adelaide O35--have been described. It has been proven that it is based partially on the similar lipopolysaccharide structures of these pathogens. In this study the involvement of the outer membrane proteins of these strains in the serological cross-reaction is presented. Microfluidic chip technology was applied for the detection of common proteins, which provided fast and quantitative data about the proteins that might be responsible for serological cross-reaction. Two outer membrane proteins with apparent molecular mass of 36 and 41 kDa, respectively, could be detected in the profile of each strain, while individual dominating protein peaks have also appeared in the protein profiles. The presence of common protein antigens was proven by Western blotting.

Genetic diversity of the O antigens of Proteus species and the development of a suspension array for molecular serotyping.

Proteus species are well-known opportunistic pathogens frequently associated with skin wound and urinary tract infections in humans and animals. O antigen diversity is important for bacteria to adapt to different hosts and environments, and has been used to identify serotypes of Proteus isolates. At present, 80 Proteus O-serotypes have been reported. Although the O antigen structures of most Proteus serotypes have been identified, the genetic features of these O antigens have not been well characterized. The O antigen gene clusters of Proteus species are located between the cpxA and secB genes. In this study, we identified 55 O antigen gene clusters of different Proteus serotypes. All clusters contain both the wzx and wzy genes and exhibit a high degree of heterogeneity. Potential functions of O antigen-related genes were proposed based on their similarity to genes in available databases. The O antigen gene clusters and structures were compared, and a number of glycosyltransferases were assigned to glycosidic linkages. In addition, an O serotype-specific suspension array was developed for detecting 31 Proteus serotypes frequently isolated from clinical specimens. To our knowledge, this is the first comprehensive report to describe the genetic features of Proteus O antigens and to develop a molecular technique to identify different Proteus serotypes.

Major involvement of bacterial components in rheumatoid arthritis and its accompanying oxidative stress, systemic inflammation and hypercoagulability.

We review the evidence that infectious agents, including those that become dormant within the host, have a major role to play in much of the etiology of rheumatoid arthritis and the inflammation that is its hallmark. This occurs in particular because they can produce cross-reactive (auto-)antigens, as well as potent inflammagens such as lipopolysaccharide that can themselves catalyze further inflammagenesis, including via ß-amyloid formation. A series of observables coexist in many chronic, inflammatory diseases as well as rheumatoid arthritis. They include iron dysregulation, hypercoagulability, anomalous morphologies of host erythrocytes, and microparticle formation. Iron dysregulation may be responsible for the periodic regrowth and resuscitation of the dormant bacteria, with concomitant inflammagen production. The present systems biology analysis benefits from the philosophical idea of "coherence," that reflects the principle that if a series of ostensibly unrelated findings are brought together into a self-consistent narrative, that narrative is thereby strengthened. As such, we provide a coherent and testable narrative for the major involvement of (often dormant) bacteria in rheumatoid arthritis.

Junctional diversification in the generation of the precursor of a discrete immune response.

Phosphocholine (PC)-specific antibodies that arise in the mouse in response to Proteus morganii (PM) and use V1-DFL16.1-JH1 are characterized by a number of recurring mutations. Most striking is an invariant A for G substitution in codon 95 of VH which results in an asparagine instead of aspartate at that position. Because of the apparent importance of this substitution in an anti-PC(PM) response, we wanted to determine the molecular basis for this base change. A cDNA library derived from pre-immune splenic B cells was examined for the frequency of VDJ containing the A substitution at 95 and the presence of additional point mutations in these sequences. Six different cDNA were isolated which contained an A substitution at the VD junction (frequency 0.00009); a seventh positive cDNA could not be examined. The V segments of four of these cDNA matched known germline genes and were, therefore, unmutated. Two others closely matched V in families whose members have not all been characterized, hence, it is not known whether the mutations observed are somatic or germline in origin. Sequences of 35 cDNA clones, containing the same V segment but differing in D, J and junctional nucleotides, revealed no mutations. These results indicate that the A substitution generated at codon 95 is most likely a product of V-DJ joining.

The antigens contributing to the serological cross-reactions of Proteus antisera with Klebsiella representatives.

Proteus sp. and Klebsiella sp. mainly cause infections of the urinary and respiratory tracts or wounds in humans. The representatives of both genera produce virulence factors like lipopolysaccharide (LPS) or outer membrane proteins (OMPs) having much in common in the structures and/or functions. To check how far this similarity is revealed in the serological cross-reactivity, the bacterial masses of 24 tested Klebsiella sp. strains were tested in ELISA with polyclonal rabbit antisera specific to the representatives of 79 Proteus O serogroups. The strongest reacting systems were selected to Western blot, where the majority of Klebsiella masses reacted in a way characteristic for electrophoretic patterns of proteins. The strongest reactions were obtained for proteins of near 67 and 40 kDa and 12.5 kDa. Mass spectrometry analysis of the proteins samples of one Proteus sp. and one Klebsiella sp. strain showed the GroEL like protein of a sequence GI number 2980926 to be similar for both strains. In Western blot some Klebsiella sp. masses reacted similarly to the homologous Proteus LPSs. The LPS contribution in the observed reactions of the high molecular-mass LPS species was confirmed for Klebsiella oxytoca 0.062.

Diagnostic criteria for scrub typhus: probability values for immunofluorescent antibody and Proteus OXK agglutinin titers.

The sensitivities and specificities of the indirect microimmunofluorescent antibody (IFA) and Weil-Felix (OXK) tests for scrub typhus were established for a range of titers using groups of diseased and control (other febrile illnesses) patients diagnosed by other methods. At a cut-off point of greater than or equal to 1:400, the IFA test was 0.96 specific, and at greater than or equal to 1:320, the OXK was 0.97 specific. Using either these highly specific levels of antibody or other rigorous diagnostic criteria (isolation or 4-fold rising titers), the prevalence of scrub typhus infection was determined to be 0.22 in an unselected population of febrile patients in a rural Malaysian hospital. Probability values (Pr) for the correct diagnosis of scrub typhus were then calculated from the specificity, sensitivity and prevalence determination for a range of titers. The Pr for an OXK titer of greater than or equal to 1:320 was 0.79, and the Pr for an IFA titer of greater than or equal to 1:400 was 0.78. When both these titers were present in a single specimen, the Pr increased to 0.96.

The haemagglutinins and fimbriae of Proteus penneri.

The haemagglutinins and fimbriae produced by 18 strains of Proteus penneri were studied and compared with those formed by representative strains of other species of Proteeae. After repeated subcultures at 30 degrees C, 12 P. penneri strains formed only MR/K haemagglutinins which were associated with thin, non-channelled, type-3 fimbriae. Two strains formed simultaneously both MS and MR/K haemagglutinins associated with thick, channelled, type-1 fimbriae and type-3 fimbriae, respectively. Four strains formed simultaneously both MR/K and MR/P haemagglutinins. No P. penneri strain formed either MS or MR/P haemagglutinins alone under these conditions. The type-3 fimbriae from P. penneri strain E180 were isolated, purified and found to be protein of 19 Kda. Immunoelectronmicroscopy studies with antibody to the type-3 fimbriae of strain E180 showed that P. penneri strains at least two antigenic types of type-3 fimbriae. The type-3 fimbrial antigen of the vaccine strain E180 was shared by another eight strains of P. penneri. A further eight P. penneri strains and the strains representative of other genera within Proteeae had type-3 fimbriae of a different antigenic type. The formation of these haemagglutinins and fimbriae suggests that this organism is well endowed to be a urinary-tract pathogen.

Fimbrial and non-fimbrial haemagglutinins in Enterobacter aerogenes.

Ten strains of Enterobacter aerogenes were examined for their ability to produce haemagglutinins and fimbriae. Nine strains formed a mannose-sensitive (MS) haemagglutinin associated with thin (4 nm) non-channelled fimbriae. These thin fimbriae of E. aerogenes were antigenically different from the thin fimbriae of other fimbriate strains of Enterobacter and Klebsiella and probably represent a new kind of fimbria not previously described in Enterobacteriaceae. Eight of these same nine strains also formed a non-fimbrial mannose-resistant, proteus-like (MR/P) haemagglutinin. The formation of thin fimbriae associated with MS haemagglutinin and of non-fimbrial MR/P haemagglutinin are properties not associated with other strains of Enterobacter and Klebsiella. E. aerogenes strain NCIB11460 was unusual among the strains examined in this series in that it alone produced mannose-resistant, Klebsiella-like (MR/K) haemagglutinin and type-3 fimbriae which, as judged by immunoelectronmicroscopy, were antigenically like those of type-3 fimbriate Klebsiella strains. The identifying characters of this exceptional strain of E. aerogenes are discussed in detail. All ten strains also produced thick fimbriae which by immunoelectronmicroscopy behaved like the type-1 fimbriae of Klebsiella strains. However, correlation between their presence and the production of MS haemagglutinin in E. aerogenes was not established. The findings are discussed in the light of the present difficult taxonomic status of E. aerogenes within the tribe Klebsielleae.

Impairment by Bacteroides species of opsonisation and phagocytosis of enterobacteria.

The ability of human polymorphonuclear leucocytes to phagocytose and kill Proteus mirabilis was impaired in vitro when the human serum, used to opsonise the target bacteria, was pretreated with cultures of various Bacteroides species. Live and dead, either heat-killed or clindamycin-treated, bacteroides cells elicited the same phenomenon. When bacteroides-treated serum was used to opsonise different Proteus species, the subsequent uptake of all strains by polymorphonuclear leucocytes was inhibited, whereas bacteroides-treated serum inhibited the uptake of some but not all of the test strains of Escherichia coli. The opsonic activity of untreated human serum was reduced when the classical complement pathway was inhibited by ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetra-acetic acid (EGTA); subsequent treatment with bacteroides did not further reduce the opsonic activity of the serum for P. mirabilis.

Immunoblot cross-reactions among Rickettsia, Proteus spp. and Legionella spp. in patients with Mediterranean spotted fever.

Sera from patients suffering from Mediterranean spotted fever (i.e. an infection due to Rickettsia conorii) were studied by immunoblot to investigate cross-reactivity. A prevalence of IgM antibodies to Proteus OX 19, Proteus OX 2, to the Rickettsia typhus group, to Legionella pneumophila serovars 4 and 5, to L. bozemanii Wiga and to L. micdadei Tatlock was found. Western blot confirmed that the antibodies were directed against the lipopolysaccharide as demonstrated by proteinase K digestion of the antigens. Cross-adsorptions showed that there is a common cross-reacting epitope among L. bozemanii Wiga, R. typhi and Proteus OX 19 but cross-reacting antibodies to L. micdadei and OX 2 were distinct and independent. This IgM cross-reaction could lead to a misdiagnosis.

Structure of the O-polysaccharide of Proteus penneri 28 and Proteus vulgaris O31 and classification of P. penneri 26 and 28 in Proteus serogroup O31.

The lipopolysaccharides (LPS) of Proteus penneri 28 and Proteus vulgaris O31 (PrK 55/57) were degraded with dilute acetic acid and structurally identical high-molecular-mass O-polysaccharides were isolated by gel-permeation chromatography. Sugar analysis and nuclear magnetic resonance (NMR) spectroscopic studies showed that both polysaccharides contain D-GlcNAc, 2-acetamido-2,6-dideoxy-L-glucose (L-2-acetamido-2,6-dideoxyglucose (N-acetylquinovosamine)) and 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-D-glucose (N-acetylisomuramic acid) and have the following structure: [carbohydrate structure: see text] where (S)-1-carboxyethyl [a residue of (S)-lactic acid] (S-Lac) is an ether-linked residue of (S)-lactic acid. The O-polysaccharide studied is structurally similar to that of P. penneri 26, which differs only in the absence of S-Lac from the GlcNAc residue. Based on the O-polysaccharide structures and serological data of the LPS, it was suggested classifying these strains in one Proteus serogroup, O31, as two subgroups: O(31a), 31b for P. penneri 28 and P. vulgaris PrK 55/57 and O31a for P. penneri 26. A serological relatedness of the LPS of Proteus O(31a), 31b and P. penneri 62 was revealed and substantiated by sharing epitope O31b, which is associated with N-acetylisomuramic acid. It was suggested that a cross-reactivity of P. penneri 28 O-antiserum with the LPS of several other P. penneri strains is due to a common epitope(s) on the LPS core.

Structure of the O-specific polysaccharide of Proteus penneri strain 41 from a new proposed serogroup O62.

O-specific polysaccharide of Proteus penneri strain 41 was studied using 1H- and 13C-NMR spectroscopy, including two-dimensional COSY, heteronuclear 13C,1H-correlation (HETCOR) and one-dimensional NOE spectroscopy, and the following structure of a non-stoichiometrically O-acetylated hexasaccharide repeating unit was established:[structure: see text] where RGlcNAc is 2-acetamido-4-O-[(S)-1-carboxyethyl]-2-deoxyglucose. Cross-reactivity of anti-P. penneri 41 O-serum with other P. penneri strains is discussed, and a new, separate O62 serogroup is proposed which is the next Proteus O-serogroup containing P. penneri strains only.

Serological characterization of Proteus penneri species novum.

Serological characterization of the first collection of the 20 Proteus penneri strains is presented. All anti-0 sera were examined in microagglutination, semi-quantitative precipitation and passive hemagglutination tests. Some P. penneri lipopolysaccharides showed strong cross-reactivity in passive hemagglutination additionally confirmed by inhibition in this test. Serological similarity between species within genus Proteus is discussed.

Structure of the O-specific polysaccharide chain and serological characterization of the Proteus penneri 62 lipopolysaccharide compared with the lipopolysaccharides of the P. penneri strains.

The chemical structure of the O-specific polysaccharide chain of Proteus penneri 62 lipopolysaccharide (LPS) containing N-acetylisomuramic acid was established using acid hydrolysis, solvolysis with anhydrous hydrogen fluoride and 1H and 13C NMR spectroscopy. Cross reactivity of the anti-O-serum P. penneri 62 with a number of other strains of the same species isolated in the USA, Canada, Germany and Poland is discussed.