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Our New BMJ website does not support IE6 please upgrade your browser to the latest version or use alternative browsers suggested below. Department of Periodontology and Oral Implantology, National Dental College and Hospital, Gulabgarh, Derabassi, Distt.
It had long been recognized that individuals who recovered from a disease developed subsequent resistance to the same. Outer membrane proteinIt was seen that transcutaneous injection of 40 kDa of outer membrane protein (OMP) inhibits co-aggregation of P.
Cysteine proteinases are the most potent antigens for vaccine development.FimbriaeThese are cell surface structure components and serve as a critical antigen. Though these results encourage us to look further into this therapy, periodontal disease, being a polygenic disease, can provide a challenge. Autogenous vaccineThese are prepared from dental plaque samples of patients with destructive periodontal diseases.Plaque samples are removed from the diseased site.
Correspondence Address:Ranjan MalhotraDepartment of Periodontology and Oral Implantology, National Dental College and Hospital, Gulabgarh, Derabassi, Distt.
A key disease landmark in this arthritis model is the production of high titers of serum GPI autoAbs, which separates the initiation phase, dependent on the adaptive immune system, from the effector phase, mostly driven by innate immune system players (Korganow et al., 1999). Because IL-17 has generally been thought of as a proinflammatory cytokine, its effect on anti-GPI titers may appear surprising on first consideration. It was recently reported that a single bacterial species that is a component of normal gut microbiota, SFB was sufficient to induce the development of SI-LP Th17 cells in mice taken from an SPF facility at the Jackson Laboratory, wherein they typically show a dearth of both this bacterium and Th17 cells (Ivanov et al., 2009). SFB are Gram positive, spore-forming obligate anaerobes that have not yet been successfully cultured in vitro (Klaasen et al., 1992).
Once generated, GPI-reactive SI-LP Th17 cells are competent to exit the gut and recirculate (Sigmundsdottir and Butcher, 2008).
The influence of microbial commensals on arthritis development in other mouse models has been variable, covering the range from inhibition to little effect to augmentation (Bjork et al., 1994 and Chervonsky, 2010). Because of the relatively high rate of discordance of human rheumatoid arthritis (RA) in monozygotic twins, the role of microbes in this disorder has been of great interest, although the conclusions have often been contentious (Edwards, 2008). More generally, commensal microbes can have a variable influence on different spontaneously developing autoimmune diseases (Chervonsky, 2010). Ankle thickness was measured with a caliper (J15 Blet micrometer) as described previously (Wu et al., 2007). Cells were collected for flow cytometry by filtering crushed spleen or thymus through a 40 ?m nylon membrane.
For T cell proliferation assays, total splenocytes (2 ? 105) in tissue-culture medium were added to 96-well plates. RNA was isolated from splenocytes via Trizol (6756) and was reverse transcribed with oligo dT priming and Superscript polymerase (Invitrogen).
For inoculation of GF mice with SFB, fecal pellets were collected from SFB-monocolonized mice with sterilized test tubes in the vinyl-isolator and were preserved frozen under dry ice until immediately before oral administration.
Bacterial genomic DNA was extracted from fresh or frozen fecal samples (within an experiment the samples were treated identically) by phenol-chloroform extraction as previously described (Ivanov et al., 2009). Simulating the above thought, it was seen that vaccines, which contain milder infectious agents when given, evoke an immune response and induce specific immunity and have successfully prevented several infectious diseases for many years, and are still being investigated for many others.
They are sterilized by heat or by immersion in iodine or formalin solution and reinjected into the same patient either locally at the site or systemically. This ongoing therapy is costly, painful and has variable prognosis, in part due to poor compliance of the patients.The elucidation of the specific bacterial etiology of periodontitis suggests that the development of specific treatment modality to target site colonization or virulence of P. Poor oral health is associated with coronary heart disease and elevated systemic inflammatory and haemostatic factors. Induction of immune response to periodontopathic bacteria and its role in pathogenesis of periodontitis. Bactericidal activity of a monoclonal antibody against a recombinant 40-kDa outer membrane protein of Porphyromonas gingivalis. Humoral immune response to antigen of porphyromonas gingivalis ATCC33277 in chronic periodontitis. Immunization with Porphyromonas gingivalis cysteine protease effects on experimental gingivitis and ligature induced periodontitis in Macaca fascicularis.
The enormity and complexity of these commensal (or mutualistic) communities have been difficult to deal with until recently, when striking advances in “next generation” sequencing methods, entailing either 16S rRNA or “shotgun” cataloguing, rendered this field navigable terrain. In both cases, ?1000 different microbial species from ?10 different divisions colonize the gastrointestinal tract, but just two bacterial divisions—the Bacteroidetes and Firmicutes—and one member of the Archaea appear to dominate, together accounting for ?98% of the 16S rRNA sequences obtained from this site. Ties to inflammatory bowel diseases are easy to understand, but the cellular and molecular mechanisms by which intestinal commensals influence autoimmune responses at distal sites remain enigmatic. Disease dampening was traced to a dearth of Th17 cells, which could be reversed by introducing segmented filamentous bacteria (SFB) into the gut of GF-housed mice, provoking rapid onset of arthritis. Gene-expression values of each group were the average values of three chips from three independent experiments.(B) Th cell signatures.
Mice were shipped from the GF Taconic facility to the SPF NYU facility on day 21 after birth and arrived the next day.
The data presented herein establish the relevance of these observations for the initiation of autoimmune disease—in particular, a nongut autoimmune disorder. Gut APCs, in particular the CD103+ subset of intestinal LP DCs, produce elevated amounts of retinoic acid, which induces associated T cells to express the gut-homing receptor, the ?4?7 integrin. They combine with circulating GPI to form immune complexes, which are deposited along the noncellular joint surface where the cartilage meets the articular cavity (Matsumoto et al., 2002).
However, the significance of these studies is difficult to assess because, in general, they relied on the administration of bacteria or bacterial products (often Complete Freunds’ Adjuvant) for the induction of disease. Most of the attention has been devoted to disease correlations with infectious microorganisms, resulting in claims of association with a number of them, including Mycobacterium tuberculosis, Proteus mirabilis, Escherichia coli, Epstein-Barr virus, retroviruses, etc. GPI peptide (GPI282-294) was added to the culture at various concentrations as indicated in the relevant figure. For microarray analysis, RNA was labeled and hybridized to GeneChip Mouse Genome 430 2.0 arrays according to the Affymetrix protocols.
Quantitative RT-PCR was performed on an Mx3000p instrument (Stratagene), with gene-specific fluorogenic assays (TaqMan, Applied Biosystems). In brief, the SI was taken, residual mesenteric fat tissue was removed, Peyer’s patches were carefully excised, and the intestine was opened longitudinally. Colonizations were performed by oral gavage with 300–400 ?l of suspension obtained by homogenizing the fecal pellets in water. Main outcome measures Primary outcomes were duration of diarrhoea and daily number and consistency of stools. Polyclonal anti-40 kDa OMP antibody exhibited potentially protective, complement-mediated bactericidal effect. Human antibody against hemagglutinin should be ideal for practical use in immunotherapy.Such treatments significantly decrease recolonization of P.
The time seems ripe to apply new, and rapidly emerging, knowledge about the composition and properties of the gastrointestinal microbiome and about the activities of recently discovered effector and regulatory T cell subsets to dissecting these mechanisms in autoimmune disease models. Thus, we provide an example of an extra-gut autoimmune disease triggered by a single member of the commensal intestinal microbiota through its promotion of a particular Th cell subset.
At later time points, the difference in anti-GPI titers under the two housing conditions was not so apparent, in contrast to the continued attenuation of ankle thickness in GF mice.
Clinical disease severity reflected the titers of GPI autoAb attained at 7 weeks of age under the three housing conditions (Figure 1D). Another instructive manner to compare gene expression in SPF and GF mice is the “volcano plots” depicted in Figure 3B, which display for each gene the SPF versus GF FC on the x axis and the p value of this FC on the y axis. Th2, Th1, and Th17 cell signatures were generated from published data sets (Nurieva et al., 2008), with 2 as the cut-off for FC over the expression value of two other cell types. In addition, when GF mice were transferred to the SPF facility, they did not succumb to arthritis if anti-IL-17 mAb was administered from the time of transfer (Figure 4B).
Sera were collected at the end of the experiment, and anti-GPI titers were quantified (right). After a 3-day rest, they were gavaged with SFB mono-feces or control GF feces (the rare animal with already swollen ankles was not used).


SFB-like bacteria have been detected morphologically in the ileum of all vertebrate species studied to date, including Homo sapiens (Klaasen et al., 1993a).
We do not rule out the possibility that other commensals can promote, or can synergize with, SFB in promoting arthritis in this model, but other species, including members of the SFB-related Clostridiaceae family, were not able to induce the accumulation of SI-LP Th17 cells in a previous set of studies (Ivanov et al., 2009).
However, SFB does not appear to operate via ATP in the SI-LP, instead upregulating the production of acute-phase isoforms of SAA in the ileum, which can act on DCs from the SI-LP to induce cocultured naive CD4+ T cells to differentiate into Th17 cells (Ivanov et al., 2009). These “gut-imprinted” T cells recirculate through the intestinal lymphatics, enter the bloodstream, and preferentially home back to the LP.
Because of the dearth of inhibitors at this site, the alternative pathway of complement is activated, leading to the recruitment and activation of inflammatory leukocytes. However, none of the associations has emerged as dominating, and mechanistic insights are lacking. And it is well known that the penetrance of type-1 diabetes in the NOD mouse strain increases with cleaner housing conditions, rising to 100% in GF facilities (Pozzilli et al., 1993). All experiments were done with protocols approved by Harvard Medical School’s Institutional Animal Care and Use Committee.
The next day, plates were washed with Milli-Q water and blocked with 150 ?l per well of tissue-culture medium (RPMI-1640, 10% fetal bovine serum, 1% nonessential amino acids, penicillin, streptomycin, glutamine) for 2 hr at 37°C. After 2 days of culture, 1 ?Ci of 3H-thymidine was added to each well, the plates were incubated overnight, and cells were harvested and the radioactivity determined by a beta counter. Forward primers (FPs) and reverse primers (RPs) were from MWG Biotech, and probes for IL-4 and IFN-? were ordered from Applied Biosystems. Recipient mouse splenocytes were isolated after 2 weeks for flow cytometric analyses of B cell reconstitution. Thus, T-cell regulation seems to affect periodontal disease and T helper cells apparently interfered with periodontal bone loss.Monoclonal antibodies have been used for passive immunization against periodontitis. The total number of genes borne by the gastrointestinal microbiome has been estimated to exceed more than a 100-fold that of the human genome (Ley et al., 2006). Such discordance between autoAb titers and the degree of ankle thickening is frequent at late time points in this model.
At 5 weeks of age, SI-LP lymphocytes (left) and splenocytes (right) were isolated, stained, and analyzed by flow cytometry. Ankle thickening was measured every day from day 27 to day 33.(B) Measurement of ankle thickness beginning on day 27. Only of late has some of the focus shifted to the potential influence of microbial commensals. It is tempting to speculate that these divergent effects might, at least in part, reflect the various diseases’ differential dependence on particular Th subsets. All GF mice were given sterilized food (NIH 31M) and water and were tested weekly to establish that they were free of aerobic and anaerobic bacteria, parasites, and fungi.
For the treatment of neonates, antibiotic-supplemented water was provided to lactating mothers. After substrate addition, titers were quantified as optical density (OD) values via an ELISA reader.
After incubation, the epithelial cell layer, containing the intraepithelial lymphocytes, was removed by intensive vortexing and passing through a 100 ?m cell strainer. The complexities in the etiopathogenesis of the periodontal diseases have been the prime obstacle in the hunt for vaccine. Passive immunization with a monoclonal antibody (61BG1.3) was shown to prevent selective colonization by P. The use of Russian probiotics preparation called ACILACT, a complex of five lyophilized lactic acid bacteria with or without "Bifidumbacterium", is claimed to improve both clinical and microbiological parameters in patients with gingivitis and periodontitis.Recently, it has been seen that inoculation of Streptococcus sanguis inhibits the growth of periopathogens, P. At this stage of disease, ankle thickness reflects primarily bone remodeling and fibrosis, and so is a cumulative index of disease duration and severity rather than an indicator of concomitant inflammation (Kouskoff et al., 1996). However, as indicated by their skewed disposition away from the right, the Th1 and Th17 cell signatures were both diminished in GF CD4+ T cells. In addition, transfer experiments showed that IL-17′s promotion of GCs was a direct effect on B cells. Data are representative of three independent experiments.(B) SI-LP lymphocytes (top) and splenocytes (middle) were isolated from SPF mice of the indicated ages, stained, and analyzed by flow cytometry, gated as indicated. Indeed, it is difficult to find any anti-GPI T and B cells in the joint itself (Kouskoff et al., 1996).
The Th1, Th2, and Th17 cell signatures were derived from the data of Dong and collaborators (Nurieva et al., 2008), each signature generated with 2 as an arbitrary FC cut-off over the expression value of the other two cell types. All experiments were done in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the New York University School of Medicine. T lymphocytes displaying the transgene-encoded TCR recognize a self-peptide derived from glucose-6-phosphate isomerase (GPI) presented by the major histocompatibility complex class II molecule, Ag7; these autoreactive T cells provide exceptionally effective help to GPI-specific B cells, resulting in massive production of GPI autoAbs, primarily of the IgG1 isotype.
Values refer to the number of genes upregulated (right) or downregulated (left) in GF vis-a-vis SPF T cells. At the end of treatment, splenocytes were isolated and stained with Abs recognizing B220, CD4, or Fas, or with PNA, and were analyzed by flow cytometry, gating as indicated.
Mean + SEM (data were a combination of two independent experiments with mice treated with metronidazole [n = 4], neomycin [n = 4], vacomycin [n = 4], ampicillin [n = 4], or nothing [n = 8]). PCR analysis of fecal material at 6 days after gavage indicated that at this early time point only those mice administered SFB-containing feces were colonized with SFB (Figure 7C).
Although the low titer of anti-GPI in the absence of SFB suffices in and of itself to explain the dampening of arthritis observed in GF-housed mice (Matsumoto et al., 1999), it remains possible that commensal microbes also impact on downstream disease processes. Cytokine transcripts in spleens were quantified by RT-PCR with hypoxanthine guanine phosphoribosyl transferase mRNA as an internal standard. However, a vaccine based on one fimbrial type may be strain specific and hence ineffective against other P. Although IL-17 is not generally thought of as a “helper” cytokine for B cells, our data are reminiscent of findings on the BXD2 model that argued that this cytokine can act on B cells by suppressing their chemotactic response to CXCL12 (Hsu et al., 2008). Clearly, this is an area that merits further exploration, which will probably need to partner with studies on animal models to establish causality, permit mechanistic dissection, and allow preclinical evaluation of suggested therapeutic strategies. Abs recognizing IL-17 and IFN-? were obtained from Biolegend and BD PharMingen, respectively. After washing, alkaline phosphatase-conjugated anti-mouse total IgG was applied, and the plates were incubated for 2 hr at 37°C. After the initial 20 min, the solution was vortexed intensely and passed through a 100 ?m cell strainer. GPI:anti-GPI immune complexes initiate a self-sustaining inflammatory response that mobilizes mast cells, neutrophils, the alternative pathway of complement, Fc? receptors, tumor necrosis factor-? (TNF-?), IL-1, etc. Indeed, antibiotics such as sulfasalazine and minocyline have been known for some time to have beneficial effects on RA progression, but underlying mechanisms remain the subject of substantial controversy (Stone et al., 2003). Foxp3 staining, Foxp3 Staining Buffer Set was obtained from eBioscience, and intracellular staining was performed according to the manufacturer’s instructions. The supernatants were passed through a 40 ?m cell strainer and the cells were resuspended in 10% DMEM medium for stimulation.
Arthritis ensues rapidly (beginning at about 4 weeks of age) and with high penetrance (close to 100%).
Last, we compared the sensitivities of the SI-LP and splenic Th17 cell compartments and of arthritis development to antibiotic treatments. Cells were run on an LSRII (BD Biosciences), and analysis was performed with FloJo (TreeStar) software.
The availability of periodontal vaccine would not only prevent or modulate the course of periodontal diseases, but also enhance the quality of life of people for whom periodontal treatment cannot be easily obtained. For many years, researchers have been exploring vaccination techniques in animal models to eradicate periodontal disease with mixed success. The plates were rinsed with water, dried overnight in the dark, and analyzed with the CTL-ImmunoSpot UV Analyzer. Conclusions Not all commercially available probiotic preparations are effective in children with acute diarrhoea. In the last decade, gene transfer research has led to a novel way to achieve vaccination as discussed below: Salivary gland of mouse, when immunized using plasmid DNA encoding the P.


Humoral immunity depends on the appearance of antibodies produced by plasma cells in the blood.
They postulated that the IgG responses to fimbriae antigen elicited by the initial contact with A. Interestingly, disease was actually exacerbated in the neomycin-treated animals, suggesting an additional negative influence of Gram-negative gut bacteria.
Cell-mediated immunity depends mainly on the development of T cells that are specifically responsive to the inducing agent and is generally active against intracellular organisms. The antibody produced by effector B lymphocytes following contact with an antigen for the first time, is usually of short duration and is characterized by a slow rise and rapid fall of immunoglobulin in the serum and this response is known as primary response.
Similarly, secreted fimbrillin in saliva could bind to pellicle components, blocking the attachment.Scientists have also demonstrated the efficacy of immunization with genetically engineered S. We evaluated the efficacy of five probiotic preparations for the treatment of acute diarrhoea in children.MethodsThe study was a prospective single blind randomised controlled trial in which parents of children with acute diarrhoea received written instructions to purchase a specific brand of probiotic. Homologues of specific stress protein families have been demonstrated to be present in oral bacteria including Fusobacterium nucleatum, Prevotella intermedia, Prevotella melaninogenica, A. The trial was performed in collaboration with family paediatricians, who in the Italian public health system care for children up to 12 years of age.
Approximately two-thirds of all the pathogens infect humans via these routes.The combined mucosal surfaces of the body comprise a considerable area of some 400 m 2 to which mouth contributes about 240 cm 2 , which must be protected from invasion by infectious agents and penetration by toxins and allergens. Eligible children were those aged 3-36 months who were seen in paediatricians' offices from October 1999 to September 2000 because of diarrhoea. Though in humans a highly sophisticated and complimentary host defense system is present, there are some microorganisms that evade the host defenses and are responsible for disease production.
The recombinant hemagglutinin B (rHagB), when injected subcutaneously in Fischer rats infected with P. We included in the study all children with diarrhoea lasting less than 48 hours for whom parents gave informed consent. For bacteria which are capable of evading host immune responses and invading the tissues P. The researchers responsible for enrolling the patients allocated the next available number on entry into the trial, and the parent of each child received written instructions to purchase the assigned probiotic product.Probiotic preparations were prescribed for five days and administered orally in 20 ml water according to the manufacturers' instructions.
All the probiotic products used in this study were available only in pharmacies and had a similar brand image and price. Table 1: Select bacterial properties involved in evasion of host defense mechanismsClick here to viewTo decrease the incidence of periodontal disease related systemic diseases It has now been well documented that periodontal diseases are not isolated lesions affecting the tooth and its supporting tissues, but have various systemic sequelae. The group of children who received only oral rehydration served as controls.The primary outcome measures were the total duration of diarrhoea and the number of stools a day and their consistency.
Duration of diarrhoea was the time in hours from the first to the last abnormal (loose or liquid) stools preceding a normal stool output.
Stool consistency was evaluated through a score system, as previously described,15 and faeces were graded as 1 (normal), 2 (loose), 3 (semiliquid), and 4 (liquid). We also investigated safety and tolerability.On enrolment we identified each child, determined the duration and severity of diarrhoea, assessed associated clinical features (fever, vomiting, dehydration), and established nutritional status and previous treatment. They were instructed to record daily the number of faecal outputs and their consistency, the type and doses of probiotic preparation taken by the child, the presence of vomiting and fever, any necessity for hospital admission, and all adverse events.The study was performed according to a multicentre single blind and controlled design.
Because of the problems of performing a double blind study of commercially available products in a large population, we used the third party blind observer method to assess efficacy. To ensure unbiased assessment, the family paediatricians, who were in charge of treatment allocation, gave written instructions to the parents to purchase a brand of probiotic and verified compliance on the reporting form, whereas the investigators collecting the reporting forms were blinded to the assigned treatment. All reporting forms were delivered to the coordinating centre at the Department of Paediatrics for analysis.
This estimate assumes a mean difference in duration of diarrhoea of 24 hours between the treated and control children (corresponding to means of 120 and 96 hours) with an SD of 30 hours within the group.
Nonhuman primates and humans are similar in both periodontal structure and microflora composition. This computation was based on the results of a preliminary open trial.15 To investigate the secondary outcomes, we doubled the number of patients. However, ligatures must be tied around the teeth to elicit periodontitis in nonhuman primates because it is difficult to colonize the oral cavity with P.
Our estimation of sample size allowed for a drop out of up to 10%.Statistical analysisA statistician blind to allocation performed the statistical analyses. We used ?2 test for categorical variables and Mann-Whitney U test for continuous variables by analysing differences between group 1 and groups 2-6.
The total duration of diarrhoea was significantly lower in children receiving Lactobacillus GG (group 2) and in those receiving the bacterial mix (group 5) than in patients receiving oral rehydration alone (group 1) (table 3)?. Since rats resemble humans in periodontal anatomy and bacterial composition, bone loss can be evaluated. Two preparations reduced the duration and severity of diarrhoea, whereas the three others had no significant effect. A formula with St thermophilus and B bifidum, two of the four bacterial species in the effective preparation, protected against diarrhoea in chronically sick children aged below 24 months.5 The three other preparations we evaluated had no or little clinical effect.
This was unexpected in the case of S boulardii because a previous controlled trial showed it to be beneficial in children admitted to hospital for diarrhoea28—that is, with a more severe condition than the mild to moderate diarrhoea in the children in our trial—which could explain the different results obtained in the two studies. A previous trial with Streptococcus faecium strain SF68 resulted in clinical improvement in children with diarrhoea associated with respiratory infection and treated with parenteral antibiotics,29 though it had no effect in adults with diarrhoea.30 Finally, the B clausii preparation had no effect. None of the preparations had a significant effect on secondary outcomes, probably because of the relatively low incidence of fever, vomiting, and hospital admissions in our children. No side effects were recorded.Diarrhoea in developed countries is usually self limiting, and active treatment is not generally recommended. All the children enrolled in our study were outpatients, and microbiological investigations were performed only in a few. Based on the findings of a large study conducted in Italy,31 it is reasonable to assume that most of the children were affected by viral acute gastroenteritis.Possible confoundingWe cannot exclude the possibility that expectations of parents confounded our results. The brands investigated were among the most widely used probiotic preparations in Italy when the study was conducted.
These products were not advertised in the press or on television, there were no remarkable differences in their brand image, and they were available only in pharmacies at the time of the study. Similarly, in Italy it is unlikely that the public would perceive one micro-organism to be more effective than another.
The lack of preconceptions about the efficacy of treatment seems to be supported by our finding that only one of the two most widely used probiotic brands in Italy was effective (group 2) whereas the other was not (group 4). A high or low cost might have affected expectations either positively or negatively, but parents were probably unaware of the comparative costs of the products investigated. From these observations, it seems unlikely that parents advised to purchase one product would have higher or lower expectations than parents assigned to purchase another product, though we cannot exclude this.In conclusion, the efficacy of probiotic preparations for the treatment of childhood acute diarrhoea is related to the individual strains of bacteria. PC, FA, LC, MIS, ADeV, and AP cared for the patients and contributed to the final version of the report.
GDeM and GT performed the data analysis and FM contributed to the design of the study, developed the computer generated randomisation list, and performed statistical analyses. Probiotics in gastrointestinal diseases in children: hard and not-so-hard evidence of efficacy. Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus.
A double-blind, placebo controlled study of the efficacy of Lactinex in the prophylaxis of amoxicillin-induced diarrhea. Biotherapeutic agents: a neglected modality for the treatment and prevention of selected intestinal and vaginal infections. Costs associated with outpatient diarrhoea in infants and toddlers: a nationwide study of the Italian Society of Paediatric Gastroenterology and Hepatology (SIGEP).



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