Longitudinal study on the effect of oral hygiene measures on the salivary count of microbial species with cariogenic potential.

The effect of oral hygiene education measures and professional tooth cleaning on the salivary levels of microbial species with high cariogenic potential (i.e. Streptococcus mutans, Lactobacillus spp. and Candida albicans) was evaluated at different time points. At time 0, high salivary carriage rates were recorded in the study group (n=30). Fifty percent of the subjects harbored all three species in their saliva, 27% harbored 2 species, and 23% only one species. At 3 months after oral hygiene measures, a statistically significant reduction was observed in salivary count of S. mutans and Lactobacillus spp. The percentage of subjects harboring all three species was also highly reduced, along with an overall improvement of clinical and risk factors parameters. At 8 months after oral hygiene measures, S. mutans and Lactobacillus spp. load was still statistically lower than that recorded at time 0, although an increment in bacterial load and a partial worsening of clinical and risk factors parameters were observed. S. mutans count in saliva inversely correlated with salivary pH, while it positively correlated with C. albicans salivary levels. The results obtained suggest that strengthening of the motivation and administration of oral hygiene instructions and professional tooth cleaning every 6-8 months, might be necessary to control salivary levels of cariogenic species.

Interaction of Mycobacterium tuberculosis cell wall components with the human natural killer cell receptors NKp44 and Toll-like receptor 2.

We have previously demonstrated that a soluble form of the human NK cell natural cytotoxicity receptor NKp44, binds to the surface of Mycobacterium tuberculosis (MTB). Herein, we investigated the interaction of MTB cell wall components (CWC) with NKp44 or with Toll-like receptor 2 (TLR2) and the role of NKp44 and TLR2 in the direct activation of NK cells upon stimulation with MTB CWC. By using several purified bacterial CWC in an ELISA, we demonstrated that NKp44 was able to bind to the MTB cell wall core mycolyl-arabinogalactan-peptidoglycan (mAGP) as well as to mycolic acids (MA) and arabinogalactan (AG), while soluble TLR2 bound to MTB peptidoglycan (PG), but not to MA or AG. The mAGP complex induced NK cell expression of CD25, CD69, NKp44 and IFN-γ production at levels comparable to M. bovis Bacillus Calmette-Guérin-stimulated (BCG) cells. While AG and MA used alone failed to induce NK cell activation, mycobacterial PG-exhibited NK cell stimulatory capacity. Activation of resting NK cells by mAGP and IFN-γ production were inhibited by anti-TLR2 MAb, but not by anti-NKp44 MAb. Differently, anti-NKp44 MAb partially inhibited CD69 expression on NK cells pre-activated with IL-2 and then stimulated with mAGP or whole BCG. Overall, these results provide evidence that components abundant in mycobacterial cell wall are able to interact with NKp44 (AG, MA) and TLR-2 (PG), respectively. While interaction of TLR2 with mycobacterial cell wall promotes activation of resting NK cells and IFN-γ production, NKp44 interaction with its putative ligands could play a secondary role in maintaining cell activation.

Human beta-defensin-3: a promising antimicrobial peptide.

The field of naturally occurring antimicrobial peptides is a research area rapidly expanding due to the high potential of such molecules as new antimicrobial drugs. In this regard, the human beta-defensin-3 is particularly attractive because of its strong antibacterial activity, relative salt-insensitiveness and low toxicity for host cells.

Complete release of adenosine deaminase from mouse lymphocytes stabilized by low-pH acetate.

Complete release of adenosine deaminase from mouse lymphocytes takes place when intact cells are stabilized by low-pH acetate buffer. Both the low pH and the acetate affect the enzyme extraction markedly. At pH 5.0 all the adenosine deaminase activity detectable in the whole cell homogenates is released into the acetate buffer in very few minutes, with a total amount of 2% protein being extracted. The complete extraction of the enzyme activity is never observed when, at pH 5.0, the acetate is replaced by glutamate, citrate, succinate or maleate and only 45% and 15% of the adenosine deaminase activity is extracted by the acetate at pH 6.0 and 7.0, respectively. The breakdown of adenosine by the enzyme activity extracted from the stabilized cells is due to deamination alone, since inosine is the only product of the catalyzed reaction and its formation is completely inhibited by coformycin, a selective inhibitor of adenosine deaminase. The enzyme extracted shows a specific activity 50-times higher than that found in the crude homogenates, and a substantial purification of the enzyme extracted is achieved by a single Sephadex G-100 gel filtration.

Questioning the role of adenosine deaminase in the development of B lymphocytes in chicken bursa.

Adenosine deaminase, a purine metabolic enzyme, was studied in lymphoid tissues of the developing chicken in order to evaluate whether enzyme activity is related to development of the immune system in birds in the same way as for mammals, in which adenosine deaminase is essential for lymphocyte differentiation, especially for the T-cell lineage. Enzyme activity was assayed in thymocytes and bursal lymphocytes at different times during chicken development ranging from the 17th day of embryonic life up to the 50th day after hatching. Adenosine deaminase activity was significantly higher in the bursa than in the thymus, regardless of whether such an activity was expressed per mg protein or per 10(8) cells; moreover, no substantial difference in the relative levels of adenosine deaminase was observed in thymocytes at the various stages of thymus development studied. Significant changes in enzyme activity, however, were found in bursal lymphocytes in which different amounts of adenosine deaminase appeared to be related to definite stages of bursal development and to specific immunological responsiveness of B lymphocytes to intravenously injected antigens. Therefore, if adenosine deaminase does play a role in the functional maturation of the immune system in birds, such a role appears to be related to the differentiation of the B- rather than the T-cell lineage.

Purine metabolism and B-lymphocyte development in the chicken bursa of fabricius.

The activity of three enzymes involved in the salvage pathway of purine nucleosides--purine nucleoside phosphorylase (PNP), xanthine dehydrogenase (XDH), and hypoxanthine-guanine phosphoribosyl transferase (HGPRT)--was investigated in cellular fractions of the chicken bursa of Fabricius differentially enriched in epithelial cells or lymphocytes. Markedly increasing levels of PNP and XDH were observed along with the enrichment in epithelial cells together with a slight, though significant, decrease in HGPRT activity. By contrast, a dramatic fall in PNP and XDH activities was detected along with the enrichment in lymphocytes together with a slight, though significant, increase in HGPRT activity. This sharply different distribution of the three enzymes, all sharing hypoxanthine as a substrate, clearly indicates that lymphocytes preferentially channel hypoxanthine into the salvage and interconversion pathways, phosphorylating it to IMP, while epithelial cells rapidly catabolize such a purine base to uric acid. Moreover, epithelial cells, unlike lymphocytes, are able to retain high intracellular levels of both hypoxanthine and inosine. These results support the possibility that epithelial cells contribute to the normal development of bursal lymphocytes by supplying such actively proliferating cells with purine rings and at the same time by preventing them from accumulating potentially toxic high levels of purine nucleotides being able to rapidly eliminate excess hypoxanthine as uric acid from the bursa environment into the bloodstream.

Identification and molecular cloning of a novel secretion antigen from Mycobacterium tuberculosis and Mycobacterium bovis BCG.

A novel protein called SA-5K was identified in Mycobacterium bovis BCG (BCG) short-term culture filtrates (CFs) by means of a recently described monoclonal antibody (mAb), L8D8. This protein had an apparent molecular mass (MM) of 5 kDa, as judged by Western blotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis in reducing conditions, and did not seem to contain any sugar or lipid substituents. In the present work, SA-5K was purified from BCG CFs by affinity chromatography. A protein that could be detected in Western blot but not by standard protein staining techniques was obtained. When SA-5K was subjected to aminoterminal sequencing, the 10 amino acids (aa) found matched the first 10-aa sequence deduced from an open reading frame (ORF) of M. tuberculosis. The ORF encodes a polypeptide, likely to include a signal for secretion, with an estimated MM of 8.3 kDa after signal peptide cleavage. The secretory nature of SA-5K was confirmed by the fact that it could only be detected in CFs, but not in other BCG subcellular fractions. After size exclusion chromatography, reactivity with mAb L8D8 was found to peak in the 45-50- and 14-16-kDa fractions. The latter MM was close to that estimated from the ORF of M. tuberculosis, implying that the 5-kDa antigen detected initially by Western blot in reducing conditions was a portion of SA-5K released after reduction of a disulphide bridge. The presence of the gene for SA-5K in BCG and its identity were confirmed by PCR (polymerase chain reaction) with specific primers and restriction analysis: the PCR product was slightly shorter in BCG than in M. tuberculosis. The gene coding for SA-5K was cloned by PCR from BCG and M. tuberculosis DNA and was expressed in Escherichia coli.

Analysis of the Mycobacterium bovis hsp60 promoter activity in recombinant Mycobacterium avium.

A clinical isolate of Mycobacterium avium was transformed with a new shuttle plasmid containing the Escherichia coli beta-galactosidase reporter gene under the control of the Mycobacterium bovis bacillus Calmette-Guérin (BCG) hsp60 promoter. beta-Galactosidase activity was assayed spectrophotometrically in bacterial homogenates of the recombinant strain (M. avium::lacZ) and used for quantification of the hsp60 promoter strength in different conditions of extra- and intracellular growth. Very low levels of beta-galactosidase were recorded during the exponential phase of in vitro growth, while they increased progressively during the late exponential and stationary phases. A significant increase in enzyme activity was also induced in exponentially growing cells by shifting the incubation temperature from 37 to 45 degrees C, but not from 37 to 42 degrees C nor from 30 to 42 degrees C. No induction of the promoter was observed by adding hydrogen peroxide to the cultures. Finally, beta-galactosidase levels were quantified during growth of M. avium::lacZ in murine macrophages. Soon after phagocytosis and, to a lesser extent at 1, 5 and 7 days after infection, increased levels of bacterial beta-galactosidase were observed indicating an increment in transcriptional activity of hsp60 promoter both at early phases of infection and during the course of intracellular growth.

Involvement of the Mycobacterium tuberculosis secreted antigen SA-5K in intracellular survival of recombinant Mycobacterium smegmatis.

A new protein (SA-5K) secreted in culture filtrates by Mycobacterium bovis, Mycobacterium tuberculosis, and few other mycobacterial species was previously identified and purified in our laboratory. In order to evaluate the putative role of SA-5K during intracellular mycobacterial growth, in the present study the SA-5K gene was cloned and expressed in Mycobacterium smegmatis, a rapid growing non-pathogenic mycobacterium which does not contain the gene for the protein. SA-5K expression in the THP-1 human macrophage cell line infected with the recombinant strain (M. smegmatis-pROL5K) was demonstrated by RT-PCR on RNA extracted from bacterial cells following 24 and 48 h of infection. Intracellular SA5K expression was associated with a higher cfu increase of M. smegmatis-pROL5K in comparison to the negative control strain (M. smegmatis recombinant for the cloning vector) (P=0.01). No significant change in SA-5K synthesis by M. smegmatis-pROL5K was observed when the recombinant strain was grown in vitro in different stress conditions such as iron deprivation, pH 4.5, presence of nitric oxide or hydrogen peroxide. The results presented in this study suggest a possible role for SA-5K in intracellular survival of recombinant M. smegmatis, though the function of the protein remains unknown.

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin.

Effective protection against Mycobacterium tuberculosis may be achieved in experimental animals by immunization with proteins secreted by tuberculous bacilli in the extracellular milieu during growth. In this study, monoclonal antibodies were raised against Mycobacterium bovis bacillus Calmette-Guérin (BCG) culture filtrate proteins or live BCG, in an attempt to identify novel mycobacterial secretion antigens: the localization of the antigens recognized by the monoclonal antibodies within the mycobacterial cell was studied and interspecies reactivity was also investigated. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Three of the monoclonal antibodies recognized proteins present only in culture filtrates, one reacted with a cytoplasmic antigen, while the remaining antibodies recognized components which were mainly associated with the cell wall and the cytoplasmic membrane. The chemical nature and possible identity of the antigens was checked. Three monoclonal antibodies are likely to react with novel mycobacterial antigens of 5, 42 and 82 kDa, respectively.

Interaction between piroxicam and azithromycin during distribution to human periodontal tissues.

BACKGROUND: Non-steroidal anti-inflammatory drugs and antibiotics are important in the prevention of infections and pain associated with periodontal surgery as well as in the adjunctive therapy of periodontal disease. In this study, patients undergoing oral surgery were treated with piroxicam and azithromycin to examine the interactions of these drugs on periodontal tissues. METHODS: Sixty-six patients were assigned to 3 groups and treated for 3 days as follows: 1) piroxicam 20 mg/day; 2) azithromycin 500 mg/day; or 3) piroxicam 20 mg/day plus azithromycin 500 mg/day. Samples of blood, saliva, gingiva, and alveolar bone were collected during surgery and at days 0.5, 2.5, 4.5, and 6.5 after last dose. Piroxicam concentrations were assayed by high-performance liquid chromatography and azithromycin concentrations by microbiological assay. RESULTS: In patients treated with piroxicam alone, the highest drug concentrations were found in plasma at each time point, but consistent piroxicam levels were also detected in gingival samples up to 4.5 days. The combined treatment with piroxicam plus azithromycin was associated with a reduction of piroxicam concentrations in periodontal tissues. In patients receiving azithromycin alone, high drug levels were measured in periodontal tissues up to 6.5 days. This distribution pattern did not vary in patients treated with piroxicam plus azithromycin. CONCLUSIONS: Treatment with piroxicam or azithromycin alone ensures a favorable distribution of these drugs into periodontal tissues. However, upon combined administration, azithromycin interferes negatively with the periodontal disposition of piroxicam. This interaction might depend on the displacement of piroxicam from acceptor sites at the level of periodontal tissues.

Reduced human beta defensin 3 in individuals with periodontal disease.

The human beta defensin 3 (hBD3) is widely expressed in the oral cavity and exerts strong antibacterial and immunomodulatory activities. Hence, we hypothesized that hBD3 could play a protective role in the maintenance of periodontal homeostasis, and that it could be found in gingival crevicular fluid (GCF) of healthy individuals and those with periodontitis at levels correlating with the degree of periodontal health. By using an ELISA assay to quantify hBD3 in GCF, we demonstrated that the peptide is present at levels easily detectable in the majority of healthy individuals, but it is drastically reduced in GCF from those with periodontitis. Furthermore, hBD3 levels inversely correlate with the severity of the disease and the degree of colonization by combinations of bacterial species with elevated periodontopathogenic potential. Both genetic factors and host/bacterial proteases released in diseased sites may be responsible for the observed low/null hBD3 levels in GCF from individuals with periodontitis.

Serological diversity demonstrable by a set of monoclonal antibodies to eight serotypes of the mutans streptococci.

A set of monoclonal antibodies were prepared by the conventional cell fusion of myeloma cells (SP2/0-Ag14) with spleen cells from BALB/c mice immunised with whole cells of a strain of mutans streptococci. Their specificities were examined against 35 reference strains of mutans streptococci, 34 reference strains of other oral streptococci and 8 reference strains of other microorganisms often inhabiting the oral cavity. Specificity was examined by enzyme immunoassay using whole cells. A total of 52 strains, consisting of 19 strains isolated in Japan, 19 strains isolated in Italy and 14 strains isolated in England, were characterised by conventional physiological and biochemical tests and then serotyped by the use of 8 monoclonal antibodies with different specificities. They were also confirmed by guanine-plus-cytosine contents of their nucleic acid and DNA-DNA hybridisation test. The results indicated that all monoclonal antibodies are useful for identification of 8 serotypes of the mutans streptococci responsible for dental caries. They also suggest the existence of more serological varieties among mutans species.

Human CD56bright and CD56dim natural killer cell subsets respond differentially to direct stimulation with Mycobacterium bovis bacillus Calmette-Guérin.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is capable of directly stimulating several effector functions of human natural killer (NK) cells in the absence of interleukin-12 and professional antigen presenting cells. To assess the contribution of two main human NK-cell subsets (CD56(dim) and CD56(bright)) to the overall in vitro NK-cell response to BCG, peripheral blood mononuclear cells depleted of nylon wool-adherent cells or purified NK cells were stimulated with live BCG. By combining intranuclear bromodeoxyuridine (BrdU) staining and analysis of CD56 marker intensity, statistically higher percentages of BrdU(+) cells were found among the CD56(bright) subset than the CD56(dim) subset after 6 days of stimulation with BCG. Similarly, evaluation of intracellular interferon-gamma (IFN-gamma) revealed that CD56(bright) cells were those mainly involved in IFN-gamma production in response to BCG. In contrast, the CD56(dim) subset contained higher levels of perforin and granzyme A, two key molecules for exocytosis-mediated cytotoxicity, than the CD56(bright) subset. Although 16-20-h stimulation with BCG did not substantially alter the expression of cytotoxic molecules by the two subsets, a decrease in perforin content was observed in the CD56(dim), but not in the CD56(bright) subset, following 4-h incubation with the NK-sensitive target K562 cell line. This decrease in perforin content correlated with the induction by BCG-stimulated NK cells, of early markers of apoptosis on target cells to a greater extent than unstimulated cells suggesting a major role for the CD56(dim) subset in cytotoxic activity in response to BCG. Taken together, these results demonstrate that CD56(bright) and CD56(dim) human NK-cell subsets exert different functional activities in response to a live bacterial pathogen.

Structural and stereospecific requirements for the nucleoside-triggered germination of Bacillus cereus spores.

A selection of adenosine analogues was tested for their ability to trigger germination of Bacillus cereus NCIB 8122 spores. The germination-inducing activity was governed by the structural properties of the sugar rather than the base moieties of the nucleosides. Among the sugar-modified analogues, only those containing a 2'-deoxy-D-ribose moiety promoted spore germination. Requirements for a specific molecular structure of the base were not clearly identified, although the highest activity was observed when substituents were inserted at position 6 of the purine ring. All the base-modified analogues, even those such as coformycin and 2'-deoxycoformycin with an expanded base ring, retained the germination-inducing activity of adenosine. However, of the two 2'-deoxycoformycin diastereoisomers characterized by an asymmetric carbon atom at position 8 of the homopurine ring, only the 8S-isomer induced germination, thus indicating that stereospecific configuration of the inducer, at least in the case of 2'-deoxycoformycin, appears to be essential for the initiation of spore germination. The differences in the germination-inducing activity of the various analogues tested were not affected significantly by spore activation at different temperatures, although the higher the activation temperature, the lower was the concentration of each analogue required for maximum germination.

Role of spore coats in the germinative response of Bacillus cereus to adenosine and its analogues.

Spores of the strain NCIB 8122 of Bacillus cereus have been depleted of coats by treatment with 0.1% sodium dodecyl sulfate--200 mM 2-mercaptoethanol--0.5 M NaCl (pH 9.6). The coat-depleted spores did not show any decrease in viability, heat resistance, refractility, dipicolinic acid content, or specific activities of several protoplastic enzymes. The germinative response of the coat-depleted spores to adenosine and several analogues thereof was found qualitatively similar to that obtained with intact spores. However, germination kinetics appeared to be affected by coat removal, since germination rate measured as loss of refractility was eight times slower even at inducer concentrations 10-fold higher than those required to promote optimal germination response of intact spores. Loss of heat resistance, on the other hand, was hardly affected by coat removal. These results suggest that, even though spore coats are not essential for the triggering reaction, they are required for a rapid evolution of the later events in the germination process.

Identification of distinct lymphocyte subsets responding to subcellular fractions of Mycobacterium bovis bacille calmette-Guérin (BCG).

In order to investigate the ability of Mycobacterium bovis BCG vaccination to induce immune responses toward different classes of mycobacterial antigens and the cell populations involved in such responses, proliferation of distinct human lymphocyte subsets from BCG-vaccinated donors in response to different subcellular fractions of BCG was analysed and compared with that of not sensitized subjects. Proliferation of different cell subsets was evaluated by flow cytometric determination of bromodeoxyuridine incorporated into DNA of dividing cells and simultaneous identification of cell surface markers. Although a certain degree of variability was observed among different donors, after 6 days of in vitro stimulation BCG-vaccinated subjects displayed, as a mean, a stronger blastogenic response to all the classes of antigens compared with non-sensitized ones. PPD, culture filtrates and membrane antigens induced a predominant proliferation of CD4+ T cells. In contrast, preparations enriched in cytosolic antigens elicited strong proliferation of gammadelta+ T cells which, as a mean, represented 55% of the proliferating cells. Although to a lesser extent, proliferation of gammadelta+ T cells was also elicited by preparations enriched in membrane and cell wall antigens. In response to the latter preparation proliferation of CD4+ T cells and CD16+/CD3- (natural killer (NK)) cells was observed, as well. In particular, cell wall antigens were found to induce significantly higher levels of proliferation of NK cells compared with all the other classes of antigens.

Depression of the immune responsiveness in mice treated with thioacetamide after antigen exposure.

The effects of the weak carcinogen thioacetamide (TAA) on the mouse immune response have been investigated. TAA administration up to 1 day after antigen priming markedly suppressed the antibody response to both T-dependent (sheep erythrocytes) and T-independent (trinitrophenyl-Ficoll) antigens; the compound was ineffective when given 3 or 4 days after immunization. A significant suppression of the in vitro lymphoproliferative response to the B-cell mitogen S. aureus Cowan I was evident from 3 to 48 h after TAA treatment. On the other hand, cell-mediated immune response to oxazolone was suppressed by TAA at each time tested, including that of challenge. The in vitro lymphoproliferative response to concanavalin A was decreased 12 h after TAA administration only, when adenosine deaminase activity within lymphocytes was increased. Furthermore, TAA is endowed with anti-inflammatory activity, as shown by the decreased footpad swelling and by evaluation of the inflammatory cell infiltration upon carrageenan injection. Taken together, these findings suggest selective TAA interaction with multiple targets within the immune system, including B- and T-lymphocytes, while non-specific cytotoxicity can be reasonably ruled out, since hematological determinations and phenotypic analysis of spleen lymphocyte subsets showed no relevant changes.

Identification, molecular cloning, and evaluation of potential use of isocitrate dehydrogenase II of Mycobacterium bovis BCG in serodiagnosis of tuberculosis.

Diagnosis of tuberculosis is time-consuming and requires infrastructures which are often not available in countries with high incidences of the disease. In the present study, an 82-kDa protein antigen was isolated by affinity chromatography and was identified by peptide mass fingerprinting as isocitrate dehydrogenase II, which is encoded by the icd2 gene of Mycobacterium bovis BCG. The icd2 gene of BCG was cloned by PCR, and the product of recombinant gene expression was purified and analyzed by two-dimensional polyacrylamide gel electrophoresis. The recombinant protein, named rICD2, was tested for its recognition by immunoglobulin G (IgG) antibodies from the sera of 16 patients with tuberculosis (TB) and 23 healthy individuals by Western blotting. The results showed that rICD2 is recognized by IgG antibodies from the sera of all TB patients tested at serum dilutions of > or = 1:640. At a serum dilution of 1:1,280, the sensitivity was 50% and the specificity was 86.9%. These results indicate that rICD2 might represent a candidate for use in a new assay for the serodiagnosis of TB.