Activation of vascular endothelial growth factor receptor-3 in macrophages restrains TLR4-NF-κB signaling and protects against endotoxin shock.

Toll-like receptors (TLRs) are critical in mediating innate immune responses against infections. However, uncontrolled TLR-triggered inflammation is associated with endotoxin shock. To better understand the homeostatic mechanisms induced by TLR4 signaling, we screened a group of key cytokines, chemokines, growth factors, and their receptors for bacteria- or LPS-induced expression. The surface vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligand VEGF-C were upregulated in macrophages. VEGFR-3 ligation by VEGF-C significantly attenuated proinflammatory cytokine production. Notably, ablation of the ligand-binding domain or tyrosine kinase activity of VEGFR-3 rendered mice more sensitive to septic shock. VEGFR-3 restrained TLR4-NF-κB activation by regulating the PI3-kinase-Akt signaling pathway and SOCS1 expression. Aside from targeting lymphatic vessels, we suggest a key role of VEGFR-3 on macrophages to prevent infections that is complicated with lymphoedema. Thus, VEGFR-3-VEGF-C signaling represents a "self-control" mechanism during antibacterial innate immunity.

The Role of Opioid Receptors in Immune System Function.

Research on the effects of opioids on immune responses was stimulated in the 1980s by the intersection of use of intravenous heroin and HIV infection, to determine if opioids were enhancing HIV progression. The majority of experiments administering opioid alkaloids (morphine and heroin) in vivo, or adding these drugs to cell cultures in vitro, showed that they were immunosuppressive. Immunosuppression was reported as down-regulation: of Natural Killer cell activity; of responses of T and B cells to mitogens; of antibody formation in vivo and in vitro; of depression of phagocytic and microbicidal activity of neutrophils and macrophages; of cytokine and chemokine production by macrophages, microglia, and astrocytes; by sensitization to various infections using animal models; and by enhanced replication of HIV in vitro. The specificity of the receptor involved in the immunosuppression was shown to be the mu opioid receptor (MOR) by using pharmacological antagonists and mice genetically deficient in MOR. Beginning with a paper published in 2005, evidence was presented that morphine is immune-stimulating via binding to MD2, a molecule associated with Toll-like Receptor 4 (TLR4), the receptor for bacterial lipopolysaccharide (LPS). This concept was pursued to implicate inflammation as a mechanism for the psychoactive effects of the opioid. This review considers the validity of this hypothesis and concludes that it is hard to sustain. The experiments demonstrating immunosuppression were carried out in vivo in rodent strains with normal levels of TLR4, or involved use of cells taken from animals that were wild-type for expression of TLR4. Since engagement of TLR4 is universally accepted to result in immune activation by up-regulation of NF-κB, if morphine were binding to TLR4, it would be predicted that opioids would have been found to be pro-inflammatory, which they were not. Further, morphine is immunosuppressive in mice with a defective TLR4 receptor. Morphine and morphine withdrawal have been shown to permit leakage of Gram-negative bacteria and LPS from the intestinal lumen. LPS is the major ligand for TLR4. It is proposed that an occult variable in experiments where morphine is being proposed to activate TLR4 is actually underlying sepsis induced by the opioid.

An interleukin-1beta (IL-1beta) single-nucleotide polymorphism at position 3954 and red complex periodontopathogens independently and additively modulate the levels of IL-1beta in diseased periodontal tissues.

Inflammatory cytokines such as interleukin-1beta (IL-1beta) are involved in the pathogenesis of periodontal diseases. A high individual variation in the levels of IL-1beta mRNA has been verified, which is possibly determined by genetic polymorphisms and/or by the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans. In this study, we investigated the role of an IL-1beta promoter single-nucleotide polymorphism at position 3954 [IL-1beta(3954) SNP] and the presence of the periodontopathogens in the determination of the IL-1beta levels in the periodontal tissues of nonsmoking chronic periodontitis (CP) patients (n = 117) and control (C) subjects (n = 175) and the possible correlations with the clinical parameters of the disease. IL-1beta(3954) SNP was investigated by restriction fragment length polymorphism, while the IL-1beta levels and the presence of the periodontopathogens were determined by real-time PCR. Similar frequencies of IL-1beta(3954) SNP were found in the C and CP groups, in spite of a trend toward a higher incidence of T alleles in the CP group. The IL-1beta(3954) SNP CT and TT genotypes, as well as P. gingivalis, T. forsythia, and T. denticola, were associated with higher IL-1beta levels and with higher values of the clinical parameters of disease severity. Concomitant analyses demonstrate that IL-1beta(3954) and the red complex periodontopathogens were found to independently and additively modulate the levels of IL-1beta in periodontal tissues. Similarly, the concurrent presence of both factors was associated with increased scores of disease severity. IL-1beta(3954) genotypes and red complex periodontopathogens, individually and additively, modulate the levels of IL-1beta in the diseased tissues of nonsmoking CP patients and, consequently, are potentially involved in the determination of the disease outcome.

Anti-lipopolysaccharide antibodies in acute appendicitis detected by ELISA.

In acute appendicitis the bowel transmissibility of the intestinal flora increases in relation to inflammation and edema formation. We can therefore observe an immunologic response in patients, which is detectable using different bacteria isolated from the normal intestinal flora. Our aim was to measure this immunologic reaction and to detect the relationship between this response and histologic types of acute appendicitis. Sera from 47 cases, comprising 38 patients suffering from appendicitis and 9 healthy controls, were examined. The sera were taken shortly before appendectomy and 14 days after operation. The antigens were lipopolysaccharides (LPS) extracted from bacteria of normal intestinal flora: Escherichia coli O21, O22, O33, O61, O68, Bacteroides fragilis and an absolute rough mutant: Shigella sonnei Re 4350. Antibodies were detected by ELISA. We showed a direct relationship between severity of appendicitis and registered antibody titer. Both aerobic and anaerobic bacteria play a role in infection in appendicitis. According to our serologic results the synergy of B. fragilis with E. coli from normal flora is more important in the initiation of inflammation, but in the perforation process the role of E. coli seems more important compared to that of B. fragilis.

A single amino-acid change between the antigenically different extracellular serine proteases V2 and B2 from Dichelobacter nodosus.

Dichelobacter nodosus (Dn), the causative organism of ovine footrot, secrets three distinct types of extracellular serine proteases which have been implicated in virulence. Southern analyses have shown that the proteases are encoded by three separate genes, and the genes encoding an acidic protease V5 and a basic protease have already been characterised from virulent Dn strain 198. The gene encoding the third protease type, as represented by acidic protease V2, was isolated from an EcoRI-BamHI library of strain 198 genomic DNA by probing with a polymerase chain reaction (PCR) fragment generated with oligodeoxyribonucleotides based on protease V2 amino acid (aa) sequences. A further clone from an RsaI library was isolated to complete the 5' region of the gene to yield an ORF of 1803 bp encoding a protein precursor of 601 aa. The acidic protease V2 gene, aprV2, shows the same precursor structure as the bprV and aprV5 genes with 72% and 69% similarity at the nucleotide (nt) level and with 73% and 69% similarity at the aa level, respectively. As monoclonal antibodies consistently distinguish the virulent (V) and benign (B) forms of this protease, the gene encoding the acidic protease B2 from benign Dn strain 305 was isolated using the PCR and characterized to investigate the molecular basis for this difference in antigenicity. A 2-bp substitution in a single codon was identified which appeared to be responsible for a change of epitope.

Affinity chromatography of antiserum to a gram negative organism.

An immunoadsorbent method is described for the purification of antibody directed against gram negative bacteria. Two anion exchange materials were compared for their ability to immobilize whole cells of Veillonella alcalescens, a gram negative oral bacterium. Purified antibody preparations were applied to the columns and subsequently eluted with various combinations of desorbing buffers. The quantity of recovered antibody was measured and its activity assayed, using a microagglutination technique. The highest levels of protein and specific antibody activity were recovered from Dowex-1-acetate columns desorbed by a combination of borate buffer pH 10 and 3 M KSCN pH 6, followed by levels of specific antibody activity obtained from a DEAE cellulose column desorbed by glycine--HC1, PBH 2.3.

Antigens for serological diagnosis of ovine footrot.

An antigen extracted from Dichelobacter nodosus with potassium thiocyanate (KSCN) is currently used in enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of ovine footrot, but the test lacks specificity in mature sheep. Other antigens were therefore evaluated for use in this test. Structural components of the cell envelope of D. nodosus including outer membrane, cytoplasmic membrane, lipopolysaccharide and pilus and extracellular proteases were purified from cultured D. nodosus while recombinant membrane proteins, protease and pilus antigens were also evaluated. Many antigenic components of D. nodosus participated in reactions in ELISA that were not specific for infection with D. nodosus and apart from pilus, none of the antigens resulted in improved specificity of the ELISA. Using a positive-negative cut-off to yield sensitivity of 70%, ELISA using pili from cultured D. nodosus serogroup A had a specificity of 98.3% compared with 89.7% for the ELISA with KSCN-extract as antigen (P < 0.001). Recombinant pili morphogenetically expressed in Pseudomonas aeruginosa were unsuitable for use in ELISA due to copurification of Pseudomonas antigens to which apparently healthy sheep directed antibodies. The application of ELISA with D. nodosus pilus as antigen in footrot control programs is discussed.

The relationship between gingivitis and the serum antibodies to the microbiota associated with periodontal disease in children with Down's syndrome.

Gingival inflammation in Down's syndrome children (DS) develops earlier and is more rapid and extensive than in non-DS children. Abnormalities in host response to the oral flora have been proposed as etiological factors of this gingival inflammation. However, the relationship between gingivitis and the host response to oral microorganisms in DS by age has not been determined. The objective of this study was to clarify this relationship. Sera were obtained from 75 DS subjects (aged 2 to 18 years) and their gingival health assessed using a modified PMA Index (M-PMA). Antibody titers to Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Treponema denticola (Td), Fusobacterium nucleatum (Fn), Selenomonas sputigena (Sel), Actinobacillus actinomycetemcomitans (Aa), and Streptococcus mitis (Mi) were determined using the micro-ELISA. DS subjects under 4 years old were found to have significantly more gingival inflammation than did normal children the same age. A significant positive correlation (r = 0.548, P < 0.0001) existed in the relationship between M-PMA score and plaque score for subjects in the G1 age group (deciduous dentition). At G1, the average antibody titers to Aa, Mi, and Fn exceeded those of the normal adult reference serum pool. In addition, IgG antibody titers to Pg, Aa, Fn, Sel, and Mi correlated significantly with the M-PMA scores in the G1 age group. There was a correlation between age (2 to 18 years) and these antibody titers. IgG antibody titers to Pg, Aa, Sel, and Mi increased significantly with increasing M-PMA score. Furthermore, the IgG antibody titers to Pg were higher (P < 0.05) in the most extensive disease group compared to the DS no-disease group. The IgG antibody titers to Pg at G3 (early puberty) were significantly higher when compared to G1 (preschool children). The IgM antibody titers to Aa at G3 were higher (P < 0.05) when compared to G1. This study suggests that colonization by Aa and Fn are closely associated with the onset of gingival inflammation in DS patients under 5 years old. Colonization by Pg, Aa, Sel, and Mi in DS appears to be associated with gingivitis at puberty.

[Skin microbiocenosis in patient with chronic dermatoses]. / Mikrotsenoz kozhi bol'nykh khronicheskimi dermatozami.

The skin microflora of patients with chronic dermatoses (atopic dermatitis and psoriasis) have been studied by the original "Bactotests" method. The data thus obtained indicate that the clinical picture of the disease is related to the severity of skin dysbacteriosis. The electron-microscopic study of 2 staphylococcal strains isolated from patients has revealed the presence of the immunoglobulin cover (capsule-like outer sheath consisting of immunoglobulins and other humoral protective factors) on the cell wall of these bacteria.

Polyclonal B cell activation, endotoxin tolerance, and limulus tests of endotoxin preparations of some periodontopathogens.

Potencies of polyclonal B-cell activation in C3H/HeN mice of Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis endotoxins were 0.36, 0.13 and 0.04, taking Salmonella abortusequi as 1.0. F. nucleatum and P. gingivalis endotoxins showed positive reactions in C3H/HeJ mice. Most activities in C3H/HeN other than that of F. nucleatum were suppressed by polymyxin B. In C3H/HeJ mice, similar inhibitions were only 60% for P. gingivalis and hardly observed with F. nucleatum. The resistances to polymyxin B could be due to protein in the endotoxins. A promoting effect of T cells added to B cells was observed only in the activity of F. nucleatum endotoxin in C3H/HeJ mice; there was no influence in other groups. Test endotoxins had nearly the same ability to produce colony stimulating factor as did references and could not produce the factor in tolerant mice. The clinical significance of tolerance is discussed. Regression lines of endotoxin doses and limulus activities of test endotoxins and Salmonella were parallel, either in specific or non-specific tests. The lines of two test groups were also parallel; values obtained by two tests were very close. These data indicate that the test endotoxins did not contain (1-3)-beta-D-glucan and elicited qualitatively similar limulus reactions to that of the reference, despite their different chemical natures. In conclusion, these test preparations had an endotoxicity similar to that of the reference and contribute to produce periodontitis through polyclonal B cell activation.

Chicken egg yolk antibody (IgY) controls Solobacterium moorei under in vitro and in vivo conditions.

Solobacterium moorei is a causative agent in diseases such as oral halitosis, bacteremia, and necrobacillosis-associated thrombophlebitis. The objective of this study was to determine the effectiveness of chicken egg yolk antibody (IgY) in controlling S. moorei. Intact S. moorei cells were used as an immunogen to immunize four White Leghorn laying hens. IgY, extracted from egg yolks obtained from these immunized hens, was purified using water dilution, two-step salt precipitation, and ultrafiltration. The purity of the IgY obtained was approximately 87.3 %. The antibody titer of the IgY was determined by enzyme-linked immunosorbent assay. The antibody titer peaked at 10,000 following the third immunization. In order to evaluate the inhibitory effects of the specific IgY, the growth of S. moorei in liquid media was measured every 12 h using a microplate reader at 600 nm. Biofilm formation of S. moorei was quantified by staining with crystal violet. The specific binding ability of IgY was further confirmed by the use of immunofluorescence and immunoelectron microscopy. Growth and biofilm formation of S. moorei were significantly (P<0.05) inhibited by 20 and 40 mg/ml specific IgY compared with the control. The specific IgY also decreased the bacterial level in the oral cavity of mice after infection with S. moorei. This study demonstrates that the growth and biofilm formation of S. moorei can be effectively inhibited by specific IgY. As a result, IgY technology may have application in the control of diseases caused by S. moorei.

Antibody response to anaerobic bacteria.

Anaerobes, especially the gram-negative non-spore-forming bacilli of the indigenous biota, are recognized as important agents of clinical infection; however, information regarding human antibodies to anaerobes is limited. Sporadic work, employing agglutination, gel diffusion, passive hemagglutination, and immunofluorescence, demonstrated antibody response to various Bacteroidaceae in patients with infections caused by Bacteroides or Fusobacterium. Because of the need for using homologous (autologous) isolates and the apparent antigenic heterogeneity of strains, there has been no general clinical application of these findings. Increased concentrations of antibodies to anaerobic intestinal organisms in chronic inflammatory bowel diseases have been found; however, it is unclear whether these are the effect or cause of these conditions. Natural antibodies to Bacteroidaceae, primarily of the IgM class, are widely distributed in normal humans; thus for serologic diagnosis IgG antibodies should be sought. Radioimmunoassays and various immunoelectorphoretic methods for detection of both antibody and antigen are presently being evaluated for diagnostic use in infections due to anaerobic bacteria.

Monoclonal antibodies against the ruminal bacterium Selenomonas ruminantium.

Monoclonal antibodies were raised against whole cells of two different strains of Selenomonas ruminantium and tested for specificity and sensitivity in immunofluorescence and enzyme-linked immunosorbent assay procedures. Species-specific and strain-specific antibodies were identified, and reactive antigens were demonstrated in solubilized cell wall extracts of S. ruminantium. A monoclonal antibody-based solid-phase immunoassay was established to quantify S. ruminantium in cultures or samples from the rumen, and this had a sensitivity of 0.01 to 0.02% from 10(7) cells. For at least one strain, the extent of antibody reaction varied depending upon the stage of bacterial growth. Antigen characterization by immunoblotting shows that monoclonal antibodies raised against two different strains of S. ruminantium reacted with the same antigen on each strain. For one strain, an additional antigen reacted with both monoclonal antibodies. In the appropriate assay, these monoclonal antibodies may have advantages over gene probes, both in speed and sensitivity, for bacterial quantification studies.

Lymphoproliferative responses to oral bacteria in humans with varying severities of periodontal disease.

We assessed in vitro the lymphocyte blastogenic resp onsess of peripheral blood lymphocytes to antigen extracts of a large battery of oral microorganisms in a population of humans with varying severities of periodontal disease. When the magnitudes and frequencies of statistically positive blastogenic responses to various oral microorganisms were compared, three general patterns emerged. The Actinomyces species proved to be potent stimulators of lymphocyte blastogenesis in most subjects tested, whereas Streptococcus sanguis, Campylobacter, and Eikenella corrodens stimulated few individuals. The response to these organisms correlated poorly with the severity of periodontal disease in the tested patients. However, several gram-negative anaerobic organisms, including Bacteroides asaccharolyticus and Treponema denticola, elicited statistically more frequent positive response in subjects with destructive periodontitis compared with patients with gingivitis. These results, taken together with recent microbiological findings, suggest that the specificity of the lymphocyte blastogenic response to antigens of oral bacteria correlates with the presence of these organisms in the subgingival microflora during various periodontal disease states.

Importance of Actinomyces and certain gram-negative anaerobic organisms in the transformation of lymphocytes from patients with periodontal disease.

Dental plaque deposits are known to be potent stimulants of lymphocyte transformation in patients with periodontal disease but not in normal subjects. Since plaque deposits consist mainly of whole bacteria, the cell walls of the most commonly found organisms in plaque were tested for their capacity to induce lymphocyte transformation. There was a direct correlation between the severity of peridontal disease and the amount of transformation induced by the cell walls of oral bacteria and by solubilized dental plaque. Cord blood leukocytes and lymphocytes from clinically normal people did not respond, which indicates that these stimulants are antigens rather than mitogens. Of the eleven bacteria tested, four members of the family Actinomycetaceae (Actinomyces viscosus, A. israelii, A. naeslundii, and Arachnia propionica), the related Propionibacterium acnes, and an anaerobic gram-negative anaerobic rod (27N). The high prevalence of the former organisms in the mature dental plaque that forms around the gingival crevice area and the potent efficacy with which they stimulate lymphocytes indicates that Actinomyces and certain gram-negative anaerobes may be important etiological agents in chronic periodontal inflammation in man.

Lipid A antibody determinations using ELISA on patients at a children's hospital: a preliminary report.

Using the enzyme-linked immunosorbent assay (ELISA), we studied the IgG and IgM antibody titers in various groups of pediatric patients (n = 81) infected with gram-negative organisms. Unlike the control group (n = 12), IgG antibodies were detected in only five (all under four months of age) of 19 children with sepsis. We assume that either the IgG antibodies are used up during the infection, or the lack of IgG antibodies results in a disposition to sepsis; the latter is more probable. Seventeen of 18 patients with urinary tract infections and proven renal involvement were IgM-positive. This indicates a permanent antigen stimulus, possibly in the form of a fixed antigen complex. Because of the heterogeneity of the groups studied, no overall statements can be made for the 93 children studied, some of whom were studied repeatedly. These children included 17 with tracheal colonization, 17 with recurrent urinary tract infections without proven renal changes and six with wound infections.

Isolation of lipoteichoic acids from Butyrivibrio fibrisolvens.

Lipoteichoic acid (LTA) and deacylated lipoteichoic acid have been isolated from the bovine-rumen Gram-negative anaerobe Butyrivibrio fibrisolvens by phenol extraction. Lipoteichoic acid (21.8 mumol phosphorus/g cells) consisted of a conventional 1, 3-phosphodiester-linked chain of glycerol phosphate units joined covalently to a glycolipid. It was not substituted with glycosyl or D-alalyl ester groups. Deacylated lipoteichoic acid (57.5 mumol phosphorus/g cells) was similar in constitution but lacked fatty acid esters. Lipoteichoic acid reacted serologically with antisera to the glycerol phosphate backbone of known lipoteichoic acids. The presence of similar teichoic acid polymers has also been demonstrated in some other strains of B. fibrisolvens and this is of significance in demonstrating that teichoic acids can occur in Gram-negative bacteria.

Helicobacter mustelae-induced gastritis and elevated gastric pH in the ferret (Mustela putorius furo).

Helicobacter mustelae has been cultured from the stomachs of ferrets with chronic gastritis; the lesions in the stomach have many of the same histological features seen in H. pylori gastritis in humans. To determine whether H. mustelae-negative ferrets with normal gastric mucosa were susceptible to colonization and whether gastritis developed after infection, four H. mustelae-negative ferrets treated with cimetidine were inoculated orally on two successive days with 3 ml (1.5 x 10(8) CFU) of H. mustelae; eight age-matched H. mustelae-negative ferrets served as controls. All four ferrets became colonized; H. mustelae persisted through week 24 of the study, as determined by positive gastric culture, tissue urease, and Warthin-Starry staining of gastric tissue. Superficial gastritis developed in the oxyntic gastric mucosa, and a full-thickness gastritis, composed primarily of lymphocytes and plasma cells plus small numbers of neutrophils and eosinophils, was present in the antrum. The inflammation was accompanied by an elevation of immunoglobulin G antibody to H. mustelae. At 4 weeks post-inoculation, the four infected (experimental) ferrets developed an elevated gastric pH (4.0 to 5.2) for 2 weeks. The eight control ferrets did not have gastritis; H. mustelae could not be demonstrated in gastric tissue via culture, nor was there an immune response to the bacteria. In ferrets, H. mustelae readily colonizes the stomach and produces a gastritis, a significant immune response, and, like H. pylori infection in humans, a transient elevated gastric pH after Helicobacter infection.

A multiple site-specific DNA-inversion model for the control of Omp1 phase and antigenic variation in Dichelobacter nodosus.

The molecular cloning and sequence analysis of four structurally variant linked genes (omp1A,B,C,D) that encode the major outer membrane protein of Dichelobacter nodosus strain VCS1001 are described. The isolation of rearranged copies of omp1A and omp1B, and the identification in the 5' regions of all four genes of short cross-over-site sequences that were similar to the Din family of cross-over-site sequences, suggested that site-specific DNA inversion was involved in omp1 rearrangement. Evidence for site-specific inversion of the 497 bp DNA fragment, which was located between the divergently orientated omp1A and omp1B genes, and which contained the promoter and 5' coding sequence of Omp1, was obtained by polymerase chain reaction-mediated amplification of inverted forms of these genes. However, to account for all of the omp1 gene copies cloned in this study, a more widespread inversion phenomenon must be involved in the rearrangement of these genes and a model for multiple site-specific DNA inversions at the omp1 locus is described. In this model the four structurally variant omp1 genes can be assembled from one of four structurally variant C-terminal coding regions and a conserved N-terminal coding region and can be expressed from a single promoter. It is postulated that this genetic capability endows D. nodosus with the ability to switch the antigenic specificity of one of its major surface proteins.

Cell surface protein antigen from Wolinella recta ATCC 33238T.

A high-molecular-weight (approximately 150,000) protein was selectively isolated by acid extraction from the cell surface of Wolinella recta and purified by negative adsorption on DEAE-cellulose and gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that this protein was found in W. recta but not in other Wolinella species, such as W. curva and W. succinogenes. Sera from patients with periodontitis reacted strongly with this protein antigen, whereas sera from healthy donors showed little or no reactivity, as determined by immunoblotting analysis. In serum, titers of immunoglobulin G antibodies to the protein antigen were significantly higher in patients with periodontitis than in periodontally healthy donors, as detected by an enzyme-linked immunosorbent assay.