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Antibodies produced in vitro in the detection of periodontal bacteria by using surface plasmon resonance analysis
Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. The Institution for Protein Environmental Affinity Surveys, PEAS Institut AB, Linköping, Sweden.
Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. The Institution for Protein Environmental Affinity Surveys, PEAS Institut AB, Linköping, Sweden.
Centre for Oral Rehabilitation, Public Dental Health Care, County Council of Östergötland, Linköping, Sweden.
Örebro University, School of Medicine, Örebro University, Sweden.
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2015 (English)In: Clinical and Experimental Dental Research, ISSN 2057-4347, Vol. 1, no 1, p. 32-44Article in journal (Refereed) Published
Abstract [en]

Porphyromonas gingivalis (P. gingivalis) is a major etiological agent associated with periodontitis. This study aims to develop antibodies to P. gingivalis in vitro for real-time detection of bacteria in clinical samples. Lymphocytes were isolated from whole blood of patient treated for periodontitis and were stimulated with P. gingivalis ATCC 33277. B-cell maturation to long-living antibody secreting-plasma cells was studied using flow cytometry and immunofluorescence staining. The antibodies developed in vitro were immobilized onto a CM-5 sensor chip of a biosensor to detect the presence of P. gingivalis in the gingival crevicular fluid of patients with periodontitis compared to periodontally healthy controls (n = 30). Surface plasmon resonance (SPR) analysis was performed to evaluate specific interactions of bacteria in samples with the immobilized antibodies. The results of SPR analysis were compared to the detection of P. gingivalis in the samples using DNA–DNA checkerboard hybridization technique. A clear and distinct change in lymphocyte morphology upon stimulation with P. gingivalis was observed. Anti-P. gingivalis antibodies secreted by CD38+ plasma cells showed the presence of all the four IgG subclasses. The results of DNA–DNA checkerboard analysis were in agreement with that of SPR analysis for the detection of P. gingivalis in patient samples. Furthermore, incubation with anti-P. gingivalis attenuated the bacterial response in SPR. The in vitro method for antibody production developed during this study could be used for an efficient real-time detection of periodontitis, and the attenuating effects of in vitro antibodies suggest their role in passive immunization to prevent periodontitis and their associated risk factors.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2015. Vol. 1, no 1, p. 32-44
Keywords [en]
Antibodies, B-cell, plasma cell, periodontitis, P. gingivalis, surface plasmon resonance
National Category
Microbiology in the medical area
Research subject
Biomedicine
Identifiers
URN: urn:nbn:se:oru:diva-46728DOI: 10.1002/cre2.6ISI: 000216889200006Scopus ID: 2-s2.0-84986898105OAI: oai:DiVA.org:oru-46728DiVA, id: diva2:873415
Funder
Knowledge FoundationAvailable from: 2015-11-24 Created: 2015-11-23 Last updated: 2018-04-07Bibliographically approved
In thesis
1. Development of novel tools for prevention and diagnosis of Porphyromonas gingivalis infection and periodontitis
Open this publication in new window or tab >>Development of novel tools for prevention and diagnosis of Porphyromonas gingivalis infection and periodontitis
2015 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Periodontitis is a chronic infectious disease causing inflammation and destruction of tooth-supporting structures eventually leading to tooth loss. Periodontitis is induced by different subgingival microorganisms in combination with an excessive and dysregulated immune-inflammatory response against this microbial challenge. The pathogenicity of the periodontal biofilm is highly dependent upon some "keystone" species of which Porphyromonas gingivalis is considered to be one of the most important pathogens. P. gingivalis expresses a broad range of virulence factors including lipopolysaccharides (LPS), fimbriae, adhesins, hemagglutinins and an array of proteolytic enzymes. Among these factors, cysteine proteases (gingipains) are of special importance both for the bacterial survival/proliferation and for the pathological outcome. Gingipains consist of arginine-specific proteases (RgpA and RgpB) and lysine-specific protease (Kgp). The major aim of this thesis was to develop and test novel methods for diagnosis and prevention of P. gingivalis infection and periodontitis. In study I, host lymphocytes were stimulated with P. gingivalis to develop antibodies in vitro and the anti-P. gingivalis antibodies were used for immunodetection in clinical samples. The in vitro method of antibody production developed during this study could be used for an efficient real-time detection of P. gingivalis infection and periodontitis, while the attenuating effects of the antibodies suggest a role in passive immunization to prevent periodontitis and its associated diseases. In study II, we have elucidated the properties and antimicrobial effects of different lactobacillus species and the two-peptide bacteriocin NC8 αβ on P. gingivalis. NC8 αβ was found to be efficient against P. gingivalis through bacterial binding followed by permeabilization of the membranes. Liposomal systems were acquired to verify membrane permeabilization by NC8 αβ. The antimicrobial activity of NC8 αβ was found to be rapid, potent and instant. In conclusion, soluble or immobilized NC8 αβ bacteriocins may be used to prevent P. gingivalis colonization and subsequent pathogenicity, and thus supplement the host immune system against invading pathogens associated with periodontitis.

Place, publisher, year, edition, pages
Örebro: Örebro University, 2015. p. 30
National Category
Microbiology in the medical area
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-46712 (URN)
Available from: 2015-11-24 Created: 2015-11-23 Last updated: 2018-01-10Bibliographically approved
2. Development of novel tools for prevention and diagnosis of Porphyromonas gingivalis infection and periodontitis
Open this publication in new window or tab >>Development of novel tools for prevention and diagnosis of Porphyromonas gingivalis infection and periodontitis
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Periodontitis is a chronic inflammatory disease caused by exaggerated host immune responses to dysregulated microbiota in dental biofilms leading to degradation of tissues and alveolar bone loss. Porphyromonas gingivalis is a major periodontal pathogen and expresses several potent virulence factors. Among these factors, arginine and lysine gingipains are of special importance, both for the bacterial survival/proliferation and the pathological outcome. The major aim of this thesis was to develop and test novel methods for diagnosis and prevention of P. gingivalis infection and periodontitis. In study I, anti-P. gingivalis antibodies were developed in vitro for immunodetection of bacteria in clinical samples using a surface plasmon resonance (SPR)-based biosensor. Specific binding of the antibodies to P. gingivalis was demonstrated in samples of patients with periodontitis and the results were validated using real-time PCR and DNA-DNA checkerboard analysis. In study II, we elucidated the properties and antimicrobial effects of different lactobacillus species and the two-peptide bacteriocin PLNC8 αβ on P. gingivalis. L. plantarum NC8 and 44048 effectively inhibited P. gingivalis growth and pure PLNC8 αβ induced bacterial lysis by damaging P. gingivalis membrane. In study III, we demonstrated that PLNC8 αβ dose-dependently induces proliferation and release of growth factors in gingival epithelial cells (GECs). Furthermore, PLNC8 αβ decreased P. gingivalis-induced cytotoxic effects in GECs but did not alter the effect of gingipains on cytokine expression. In study IV, we elucidated the effects of anti-P. gingivalis antibodies and PLNC8 αβ in regulating cellular responses during P. gingivalis infection. Both antibodies and PLNC8 αβ modulated P. gingivalis-induced expression of growth factors in GECs, however, their effects were diminished when used in combination. The results of this thesis demonstrate a possible role of anti-P. gingivalis antibodies and PLNC8 αβ in prevention and treatment of P. gingivalis infection and periodontitis with no cytotoxic effects on human cells.

Place, publisher, year, edition, pages
Örebro: Örebro university, 2016. p. 57
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 151
Keywords
Periodontitis, Porphyromonas gingivalis, anti-P. gingivalis antibodies, surface plasmon resonance, PLNC8 αβ, proliferation, growth factors.
National Category
Other Basic Medicine
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-52056 (URN)978-91-7668-162-8 (ISBN)
Public defence
2016-10-28, Campus USÖ, hörsal C1, Södra Grev Rosengatan 30, Örebro, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2016-09-08 Created: 2016-09-08 Last updated: 2018-01-10Bibliographically approved

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Nakka, Sravya SowdaminiLönn, JohannaBengtsson, Torbjörn

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Nakka, Sravya SowdaminiLönn, JohannaBengtsson, Torbjörn
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School of Health and Medical Sciences, Örebro University, SwedenSchool of Medicine, Örebro University, Sweden
Microbiology in the medical area

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