Antimicrobial and Anti-Inflammatory Lingonberry Mouthwash—A Clinical Pilot Study in the Oral Cavity

Fermented lingonberry juice was designed to be used as a mouthwash. Our aim was to study the antimicrobial and anti-inflammatory effects of the mouthwash in the oral cavity. A clinical study of 30 adult participants was performed. A total of 20 participants used 10 mL of the mouthwash twice daily for two weeks and 10 participants used 20 mL twice daily for one week. Streptococcus mutans, Candida and Lactobacilli were cultivated at the beginning, after the mouthwash period and after a washout period. At the same timepoints an additional oral mouthrinse was collected for chair-side/point-of-care (POC)-PerioSafe®/OraLyzer® aMMP-8 quantitative on-line evaluation, and an oral clinical investigation was performed. Mean Streptococcus mutans and Candida counts, visible plaque index (VPI) and bleeding on probing (BOP) were reduced, and Lactobacilli counts increased during the lingonberry mouthwash period. The aMMP-8 mouthrinses showed reduced values in both test groups when compared to the startpoint. The mouthrinse aMMP-8 reduction correlated with the reductions in microbial counts, VPI and BOP. Based on the results, fermented lingonberry juice seems a promising aid in oral homecare, diminishing the microbial and related proinflammatory burden by balancing the oral microbial flora and gradually lowering the inflammatory load in the oral cavity

Candida proteinases in the degradation of oral mucosal tissue components associated with Candida invasion

The aim of this thesis was to compare the degradation of human oral epithelial proteins by proteinases of different Candida yeast species. We focused on proteins associated with Candida invasion in the cell-to-cell junction, the basement membrane zone, the extracellular matrix, and local tissue inflammatory regulators. Another main objective was to evaluate the effect of the yeast/hyphal transition and pH on the degradative capability of Candida. The enzymatic activity of the Candida proteinases was verified by gelatin zymography. Laminins-332 (Lm-322) and -511(Lm-511) produced by human oral keratinocytes were gathered from the growth media, and E-cadherin (E-Cad) was isolated from the cell membrane of the keratinocytes by immunoprecipitation. The proteins were incubated with Candida cells and cell-free fractions, and degradation was detected by fluorography. Fibronectin degradation was visualised by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Matrix metalloproteinase-9 (MMP-9) activation and tissue inhibitor of metalloproteinase-1 (TIMP-1) fragmentation was detected by using the Western blot and enhanced chemoluminescence (ECL) techniques. Residual activity of TIMP-1 was evaluated by a casein degradation assay. A fluorimetric assay was used to detect and compare Candida proteinase activities with MMP-9. These studies showed that the ability of the different Candida yeast species to degrade human Lm-332, fibronectin, and E-Cad vary from strain to strain and that this degradation is pH-dependent. This indicates that local acidic pH in tissue may play a role in tissue destruction by activating Candida proteinases and aid invasion of Candida into deeper tissue. A potential correlation exists between the morphological form of the yeasts and the degradative ability; the C. albicans yeast form seems to be related to superficial infections, and hyphal forms can apparently invade deeper tissues between the epithelial cells by degradation of E-Cad. Basement membrane degradation is possible, especially in the junctional epithelium, which contains only Lm-332 as a structural component. Local tissue host inflammatory mediators, such as MMP-9, were activated, and TIMP-1 was degraded by certain Candida species, thus indicating the possibility of a weakened host tissue defence mechanism in vivo.Candida-hiivasienitulehdukset ovat yleistyvä ongelma väestön ikääntyessä immunivastetta heikentävien tautien ja lääkkeiden suuta kuivattavien sivuvaikutusten vuoksi. Candida- hiivasienten aiheuttamat tulehdukset ovat kolmanneksi yleisin mikrobitauti sairaalapotilailla ja lääkeresistentit hiivasienikannat ovat hälyyttävästi lisääntymässä. Selkeää kuvaa hiivasieni-infektion etenemisestä ei ole. Näiden seikkojen vuoksi onkin tärkeää, että tutkimusten avulla selvitetään mitkä tekijät vaikuttavat tulehduksen etenemiseen.Tähän väitöskirjatyöhön sisältyvien tutkimusten päämääränä oli selvittää voivatko Candida-hiivasienten entsyymit hajottaa suun limakalvon proteiineja ja aiheuttaa tällä mekanismilla tulehduksen etenemisen syvemmälle kudoksiin. Laboratoriotutkimuksissa testasimme seitsemää eri ienkudoksessa esiintyvää proteiinia. Näihin kuuluivat: epiteelin rakenneproteiinit laminiini-332, laminiini-511, fibronektiini ja E- kadheriini sekä matriksin metalloproteaasi-9 (MMP-9), metalloproteaasien toimintaa estävät proteiinit TIMP-1 ja TIMP-2. Candida- hiivasienikannoiksi valittiin yleisimmin esiintyvä hiivalaji Candida albicans sekä nykyisin yhä useammin tulehduksia aiheuttavia hiivakantoja, joihin kuuluvat Candida dubliniensis, Candida glabrata, Candida parapsilosis, Candida guilliermondii, Candida krusei ja Candida tropicalis. Lisäksi testasimme geneettisesti muunneltuja hiivakantoja. Saatujen tulosten perusteella voidaan todeta, että useimmat Candida- hiivakannat pystyvät hajottamaan suun limakalvon proteiineja. Näyttää mahdolliselta, että Candidat voivat edetä ientaskun epiteelin liitosepiteelin läpi tai epiteelisolujen välistä kudoksiin. Tietyt Candida-hiivakannat voivat aktivoida ienkudoksessa olevia MMP-9 entsyymejä joko suoraan tai välillisesti hajottamalla niitä estäviä proteiineja ja voivat näin edistää ientulehduksen etenemistä. Tutkimustulosten perusteella on mahdollista kehittää fluoresenssiin perustuva diagnostinen testi hiivatulehduksen toteamiseksi

Combining biochemistry to dentistry : from in vitro Candida glabrata observations to an in vivo clinical lingonberry application

Our studies focused on using Candida glabrata (C. glabrata) as a model organism to isolate and investigate the role of C. glabrata cell wall proteases as host protein- degrading virulence factors and the inhibition of their action. The cell wall proteins of microbes are in the frontline of first contact with the host cells in oral mucosa. C. glabrata is the second most prominent Candida yeast, and it is commonly found in the normal oral microbial flora causing opportunistic yeast infections, particularly in hospitalized patients. It is considered innately azole- resistant and treatment is more difficult compared to the typical Candida albicans (C. albicans) infections. Azoles are the most commonly used antifungal agents used in candidosis. There is an urgent need of development of topical antimicrobial agents, and wild berries such as lingonberry, have been increasingly studied. Lingonberries are known to have antioxidant, anti-inflammatory, antimicrobial and anticancerous properties and are considered beneficial to health. To this background we studied in vitro and in vivo the effects of a patented, fermented lingonberry juice (Lingora®, from now on abbreviated as FLJ). It was specially developed to be used as a mouthwash on C. glabrata and other typical microbes of the oral flora related to yeast infections and caries. Our primary goal was to isolate, identify and characterize C. glabrata cell wall proteases with biochemical methods: enzymatic treatment of C. glabrata cells, MDPF-zymography, SDS-PAGE, 2D-PAGE and LC-MS/MS. These methods may be used to isolate and identify novel Candida cell wall proteases enabling their further characterization and inhibition studies. Further in vitro studies were conducted on the effect of FLJ on intracellular protein expression of C. glabrata with the 2D-DIGE method. The proteins were identified by LC-MS/MS. The inhibition of proliferation and invasion of two aggressive oral tongue squamous cell carcinoma (OTSCC) lines (HSC-3, SCC-25) with FLJ and curcumin were measured in vitro by colorimetric ELISA and three- dimensional Myogel spheroid assay. Finally, we conducted a clinical pilot study including oral examinations, microbial cultivations and measurements of active MMP-8 concentrations using PerioSafe® point-of-care test. FLJ was used as a mouthwash to see if it has also in vivo effects on three microbes of the oral microbiota. From the C. glabrata cell wall we identified a novel, uncharacterized 25 kDa serine protease, Cwp1.2., with an estimated pI of 7.6 and gelatinolytic activity. This activity was inhibited by PMSF, a known serine protease inhibitor. Certain C. glabrata intracellular protein expressions related to glycolysis, oxidative phosphorylation, oxidative stress and biofilm formation were significantly diminished after treatment with FLJ. These proteins include e.g. heat shock protein and redoxin, which are expressed by C. glabrata when predisposed to stress. Downregulation of these proteins causes C. glabrata cells to be more vulnerable to environmental stress and may cause lower virulence. FLJ showed to inhibit proliferation and invasion of two aggressive OTSCC cell lines similar to curcumin. FLJ is safe, has no known interactions with medications and could be studied to be used as an adjunctive therapy in management of OTSCC. The clinical mouthwash pilot study with FLJ results showed statistically significant reduction in Candida and S. mutans counts. Our in vitro studies also indicate growth inhibitory effect on the most common periodontitis- related bacteria. Bleeding on probing (BOP), visible plaque index (VPI) and trend of active matrix metalloprotease-8 (aMMP-8) values were also reduced during the FLJ mouthwash period. Lactobacilli counts increased during the mouthwash period. Although lactobacilli are thought to be related to caries the clinical parameters and clinical outcome indicate a balancing effect on the oral microbial flora from a dysbiotic to a symbiotic direction. This diminished microbial related inflammatory burden should be studied further in context with broader positive general health effects. The results show several beneficial aspects of FLJ in the oral environment. The methodology used in these studies might be applicable to other oral microbes in developing novel antimicrobial agents related to cell wall proteases of Candida. Combined in vitro and in vivo studies showed effects of FLJ on C. glabrata intracellular proteins, host cell derived proteins including anti-inflammatory effects, tongue carcinoma cells and oral microbiota.Suun paikallishoitona käytettäviä aineita on rajatusti ja nykytutkimus on laajentunut luonnonmarjojen tutkimiseen. Marjojen katsotaan olevan osa terveellistä ruokavaliota. Puolukalla tiedetään olevan antioksidatiivisia, anti-inflammatorisia, antimikrobiaalisia ja tiettyjä syöpiä ehkäiseviä ominaisuuksia, mutta sen vaikutuksia suuhun ei ole tutkittu. Suukäyttöön soveltuvaksi puolukkamehusta poistettiin patentoidulla menetelmällä valtaosa luontaisista sokereista fermentoimalla (Lingora®). Sen vaikutuksia tutkittiin sekä laboratoriokokeilla että kliinisesti suun hiivasienitulehduksissa esiintyvien Candida- lajien ja karieksessa esiintyvien mikrobien (Streptococcus mutans, laktobasillit) kasvua ehkäisevänä aineena. Lisäksi laboratoriokokeessa tutkittiin fermentoidun puolukkamehun vaikutusta kahden aggressiivisen kielisyöpäsolulinjan jakaantumiseen ja leviämiseen. Candida glabrata (C. glabrata) on toiseksi yleisin suun mikrobiomissa esiintyvä Candida- hiivasienilaji, joka aiheuttaa vaikeita tulehduksia varsinkin sairaalapotilailla. Mikrobien soluseinä on kontaktissa suun limakalvon soluihin ja yksi tutkimusten tavoite olikin käyttää C. glabrataa malliorganismina ja tunnistaa uusia C. glabratan soluseinän proteaaseja sekä tutkia fermentoidun puolukkamehun vaikutusta C. glabratan solunsisäisten proteiinien määrään, joilla saattaa olla merkitystä sen taudinaiheuttamiskyvylle. C. glabratan soluseinästä löydettiin uusi proteaasi, jota tutkimalla on mahdollista kehittää uusia hiivasienilääkkeitä. Tutkimuksissa käytettyjä menetelmiä voi soveltaa proteaasien tutkimukseen muillakin mikrobeilla uusien antimikrobiaalisten aineiden kehittämisessä. C. glabratan solunsisäiseen aineenvaihduntaan ja stressinsietoon liittyvien proteiinien määrät vähenivät fermentoidulla puolukkamehulla. Tällä on hiivasienten kasvua sekä mahdollisesti taudinaiheuttamiskykyä heikentävä vaikutus. Laboratoriokokeissa fermentoitu puolukkamehu esti myös merkittävästi kielisyöpäsolujen kasvua ja leviämistä. Kliinisessä kokeessa fermentoitu puolukkamehu esti merkittävästi Candida- hiivasienten ja Streptococcus mutans- bakteerien kasvua. Laktobasillien määrä lisääntyi, jonka katsotaan olevan hyödyllistä yleisterveydelle. Ienverenvuoto (BOP) ja näkyvän plakin määrä (VPI) vähenivät myös ja aMMP-8 määrässä näkyi laskeva trendi. Jatkotutkimukset ovat meneillään. Fermentoitu puolukkamehu on turvallinen, eikä sillä ole tunnettuja merkittäviä lääkeaineinteraktioita. Nämä laboratorio- ja kliinisen kokeen tulokset osoittavat sillä olevan useita hyödyllisiä vaikutuksia ja sitä voidaan käyttää suun kotihoidon tukena

The Effect of Fermented Lingonberry Juice on Candida glabrata Intracellular Protein Expression

M1 - GenericPeer reviewe

Lingonberries—General and Oral Effects on the Microbiome and Inflammation

Lingonberry (Vaccinium vitis ideae L.) is a low-bush wild plant found in the northern hemisphere. The berries are used in traditional medicine in Finland to treat oral yeast infections. General and oral effects of lingonberries on the microbiome and inflammation are reviewed. A brief introduction to oral microbiome symbiosis and dysbiosis, innate and adaptive immunity and inflammation are included, and special features in microbe/host interactions in the oral environment are considered. In vitro anticancer, antimicrobial, antioxidant, anti-inflammatory, and in vivo mouse and human studies are included, focusing on the symbiotic effect of lingonberries on oral and general health

Lingonberry polyphenols : Potential SARS-CoV-2 inhibitors as nutraceutical tools?

Proposed pathway of the effect of lingonberry polyphenols on oral microbial (viral) load reduction and consequent beneficial local and systemic (respiratory tract) anti-inflammatory and antimicrobial/antiviral effects.Non peer reviewe

Effects of Fermented Lingonberry Juice Mouthwash on Salivary Parameters—A One-Year Prospective Human Intervention Study

A one-year prospective human intervention study was performed to examine the effects of fermented lingonberry juice (FLJ), used as a mouthwash for six months, on salivary parameters. A total of 25 adult participants used 10 mL of FLJ as mouthwash 30 s daily for 6 months in addition to their normal oral homecare routines. Standard oral examinations and gathering of samples were performed at the beginning of the study and after six months and one year. Resting and stimulated saliva secretion rates, resting saliva pH, and stimulated saliva buffering capacity were determined. A questionnaire of participants’ subjective sensations of mouth dryness was also recorded at each timepoint. Fermented lingonberry juice mouthwash had positive effect to all five salivary parameters and were, according to the omnibus test, statistically significant during the study period. Analysis of the subjective dry mouth sensation questionnaires revealed that symptoms of xerostomia decreased due to the use of FLJ. This study revealed that the once-a-day use of FLJ mouthwash had a beneficial, increasing effect on salivary flow rates, buffering capacity, and salivary pH. FLJ thus can be safely used as an adjunctive and beneficial therapy in oral homecare, protecting teeth and oral mucosa, including periodontium, and also relieving dry mouth symptoms

Host-Modulation Therapy and Chair-Side Diagnostics in the Treatment of Peri-Implantitis

Previous studies report periodontitis and peri-implantitis being able to induce systemic low-grade inflammation, which is known to be associated with increased risk for some systemic medical disease such as cardiovascular disease. In this regard, recent studies have shown that host modulation therapy (HMT) together with traditional mechanical and surgical treatment not only cease the progression of periodontitis but also reduce the systemic collagenolytic biomarkers in both oral fluids and circulation. This suggests that the corresponding adjunctive HMT-medication could be effective in the prevention and treatment of dental peri-implantitis, as well. Furthermore, low-cost, safe, and practical oral fluid active matrix metalloproteinase-8 (aMMP-8) lateral-flow immunotests have been proposed as point-of-care/chair-side diagnostic tools to detect peri-implantitis and periodontitis, and to monitor their effective resolutions, while using various therapeutic strategies, including host modulation. This study reports the potential benefits of HMT-medication in the prevention and treatment of dental peri-implantitis among five patients (four of five were current/ex-smokers). In addition, the aMMP-8 point-of-care test diagnosed 20 peri-implantitis and 20 healthy controls correctly. In conclusion, this study and previous studies support the potential effectiveness of HMT-medication(s) and point-of-care/chair-side technologies in the treatment and diagnostics/monitoring of peri-implantitis. However, more studies are needed to further confirm this.Peer reviewe

Fermented lingonberry juice's effects on active MMP-8 (aMMP-8), bleeding on probing (BOP), and visible plaque index (VPI) in dental implants-A clinical pilot mouthwash study

Objectives: We aimed to study the effects of fermented lingonberry juice (FLJ) as a mouthwash on the levels of active matrix metalloproteinase-8 (aMMP-8) in peri-implant sulcular fluid (PISF), bleeding on probing (BOP), and visible plaque index (VPI). We hypothesized that FU rinsing could reduce inflammation (aMMP-8 and BOP) and microbial load (VPI) in the oral cavity, especially around dental implants. Materials and Methods: A clinical pilot study was performed using FLJ as a mouthwash. The inclusion criteria were at least one dental implant in the anterior or posterior areas with a screw-retained crown. Ten participants used 10 ml of mouthwash twice a day for 15 days, and 10 participants served as the control group. Point-of-care tests (POCTs) were used to measure aMMP-8 levels in the PISF, and BOP and VPI were recorded at the beginning of the trial and after 15 and 30 days. Results: The FLJ mouthwash had a reductive effect on aMMP-8, VPI, and BOP in the mouthwash group; however, there was no significant difference compared to the control group. The difference in VPI and BOP levels between the groups diminished after the lingonberry regimen ended. The decrease in aMMP-8 levels appeared to continue even after discontinuation of the mouthwash regimen. Conclusion: The reduction in the amount of plaque, aMMP-8, arid BOP by FLJ was promising and continuous considering the relatively short study period and sample size. FLJ is a natural and safe supplement for oral and dental implant home care. Further studies are required to verify these promising results.Peer reviewe

Lingonberries—General and Oral Effects on the Microbiome and Inflammation

Lingonberry (Vaccinium vitis ideae L.) is a low-bush wild plant found in the northern hemisphere. The berries are used in traditional medicine in Finland to treat oral yeast infections. General and oral effects of lingonberries on the microbiome and inflammation are reviewed. A brief introduction to oral microbiome symbiosis and dysbiosis, innate and adaptive immunity and inflammation are included, and special features in microbe/host interactions in the oral environment are considered. In vitro anticancer, antimicrobial, antioxidant, anti-inflammatory, and in vivo mouse and human studies are included, focusing on the symbiotic effect of lingonberries on oral and general health