Identification of a new genetic variant (G231N, E232T, N235D) of peptidylarginine deiminase from P. gingivalis in advanced periodontitis.

Chronic periodontitis (CP), an inflammatory disease of periodontal tissues driven by a dysbiotic subgingival bacterial biofilm, is also associated with several systemic diseases, including rheumatoid arthritis (RA). Porphyromonas gingivalis, one of the bacterial species implicated in CP as a keystone pathogen produces peptidyl arginine deiminase (PPAD) that citrullinates C-terminal arginine residues in proteins and peptides. Autoimmunity to citrullinated epitopes is crucial in RA, hence PPAD activity is considered a possible mechanistic link between CP and RA. Here we determined the PPAD enzymatic activity produced by clinical isolates of P. gingivalis, sequenced the ppad gene, and correlated the results with clinical determinants of CP in patients from whom the bacteria were isolated. The analysis revealed variations in PPAD activity and genetic diversity of the ppad gene in clinical P. gingivalis isolates. Interestingly, the severity of CP was correlated with a higher level of PPAD activity that was associated with the presence of a triple mutation (G231N, E232T, N235D) in PPAD in comparison to W83 and ATCC 33277 type strains. The relation between mutations and enhanced activity was verified by directed mutagenesis which showed that all three amino acid residue substitutions must be introduced into PPAD expressed by the type strains to obtain the super-active enzyme. Cumulatively, these results may lead to the development of novel prognostic tools to assess the progress of CP in the context of associated RA by analyzing the ppad genotype in CP patients infected with P. gingivalis.

Principles and Limitations of miRNA Purification and Analysis in Whole Blood Collected during Ablation Procedure from Patients with Atrial Fibrillation.

Background: MicroRNA (miRNA) have the potential to be non-invasive and attractive biomarkers for a vast number of diseases and clinical conditions; however, a reliable analysis of miRNA expression in blood samples meets a number of methodological challenges. In this report, we presented and discussed, specifically, the principles and limitations of miRNA purification and analysis in blood plasma samples collected from the left atrium during an ablation procedure on patients with atrial fibrillation (AF). Materials and Methods: Consecutive patients hospitalized in the First Department of Cardiology for pulmonary vein ablation were included in this study (11 with diagnosed paroxysmal AF, 14 with persistent AF, and 5 without AF hospitalized for left-sided WPW ablation-control group). Whole blood samples were collected from the left atrium after transseptal puncture during the ablation procedure of AF patients. Analysis of the set of miRNA molecules was performed in blood plasma samples using the MIHS-113ZF-12 kit and miScript microRNA PCR Array Human Cardiovascular Disease. Results: The miRNS concentrations were in the following ranges: paroxysmal AF: 7-23.1 ng/µL; persistent AF: 4.9-66.8 ng/µL; controls: 6.3-10.6 ng/µL. The low A260/280 ratio indicated the protein contamination and the low A260/A230 absorbance ratio suggested the contamination by hydrocarbons. Spectrophotometric measurements also indicated low concentration of nucleic acids (<10 ng/µL). Further steps of analysis revealed that the concentration of cDNA after the Real-Time PCR (using the PAXgene RNA Blood kit) reaction was higher (148.8 ng/µL vs. 68.4 ng/µL) and the obtained absorbance ratios (A260/A280 = 2.24 and A260/A230 = 2.23) indicated adequate RNA purity. Conclusions: Although developments in miRNA sequencing and isolation technology have improved, detection of plasma-based miRNA, low RNA content, and sequencing bias introduced during library preparation remain challenging in patients with AF. The measurement of the quantity and quality of the RNA obtained is crucial for the interpretation of an efficient RNA isolation.

The Association of ADAMTS7 Gene Polymorphisms with the Risk of Coronary Artery Disease Occurrence and Cardiovascular Survival in the Polish Population: A Case-Control and a Prospective Cohort Study.

The aim of this study was to investigate whether the polymorphisms of the ADAMTS7 gene affect the risk of occurrence and mortality due to CAD. The study group included 231 patients diagnosed with CAD and 240 control blood donors. The genotyping of specified polymorphisms, i.e., rs1994016, rs3825807, and rs7173743, was performed using the TaqMan-PCR. We found that the C allele carriers of the rs1994016 and A allele carriers of the rs3825807 polymorphisms increased the risk of CAD, respectively: OR = 1.72, p = 0.036; OR = 1.64, p = 0.04. Moreover, we studied the biological interactions of specified variants, i.e., rs3825807, rs1994016, and rs7173743, and previously approved risk factors of CAD. We demonstrated here that selected polymorphisms of ADAMTS7 increased the risk of CAD altogether with abnormalities of total cholesterol and LDL concentrations in serum. Although survival analyses did not reveal statistical significance, we observed a trend for the AA genotype of the rs3825807 ADAMTS7, which may predispose to death due to CAD in a 5-year follow-up. In conclusion, the ADAMTS7 polymorphisms investigated in this study may increase the risk of occurrence and/or death due to CAD in the Polish population.

Analysis of miRNAs in Osteogenesis imperfecta Caused by Mutations in COL1A1 and COL1A2: Insights into Molecular Mechanisms and Potential Therapeutic Targets.

Osteogenesis imperfecta (OI) is a group of connective tissue disorders leading to abnormal bone formation, mainly due to mutations in genes encoding collagen type I (Col I). Osteogenesis is regulated by a number of molecules, including microRNAs (miRNAs), indicating their potential as targets for OI therapy. The goal of this study was to identify and analyze the expression profiles of miRNAs involved in bone extracellular matrix (ECM) regulation in patients diagnosed with OI type I caused by mutations in COL1A1 or COL1A2. Primary skin fibroblast cultures were used for DNA purification and sequence analysis, followed by analysis of miRNA expression. Sequencing analysis revealed mutations of the COL1A1 or COL1A2 genes in all OI patients, including four previously unreported. Amongst the 40 miRNAs analyzed, 9 were identified exclusively in OI cells and 26 in both OI patients and the controls. In the latter case, the expression of six miRNAs (hsa-miR-10b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, has-miR-204-5p, has-miR-216a-5p, and hsa-miR-449a) increased, while four (hsa-miR-129-5p, hsa-miR-199b-5p, hsa-miR-664a-5p, and hsa-miR-30a-5p) decreased significantly in OI cells in comparison to their expression in the control cells. The identified mutations and miRNA expression profiles shed light on the intricate processes governing bone formation and ECM regulation, paving the way for further research and potential therapeutic advancements in OI and other genetic diseases related to bone abnormality management.

Gene-repaired iPS cells as novel approach for patient with osteogenesis imperfecta.

Introduction: The benefits of patient's specific cell/gene therapy have been reported in relation to numerous genetic related disorders including osteogenesis imperfecta (OI). In osteogenesis imperfecta particularly also a drug therapy based on the administration of bisphosphonates partially helped to ease the symptoms. Methods: In this controlled trial, fibroblasts derived from patient diagnosed with OI type II have been successfully reprogrammed into induced Pluripotent Stem cells (iPSCs) using Yamanaka factors. Those cells were subjected to repair mutations found in the COL1A1 gene using homologous recombination (HR) approach facilitated with star polymer (STAR) as a carrier of the genetic material. Results: Delivery of the correct linear DNA fragment to the osteogenesis imperfecta patient's cells resulted in the repair of the DNA mutation with an 84% success rate. IPSCs showed 87% viability after STAR treatment and 82% with its polyplex. Discussion: The use of novel polymer Poly[N,N-Dimethylaminoethyl Methacrylate-co-Hydroxyl-Bearing Oligo(Ethylene Glycol) Methacrylate] Arms (P(DMAEMA-co-OEGMA-OH) with star-like structure has been shown as an efficient tool for nucleic acids delivery into cells (Funded by National Science Centre, Contract No. UMO-2020/37/N/NZ2/01125).

Pulp Revascularization After Autotransplantation of the Mandibular Canines With Partially Resected Roots: Report of 5 Cases With Follow-ups Between 26 and 80 Months.

INTRODUCTION: Transmigrated (migrated through the midline) mandibular canines constitute a treatment challenge. Advanced transmigration can be successfully treated by autotransplantation. In developing canines, pulp revascularization is typical after transplantation. On the contrary, the pulp of teeth with completed apex formation does not undergo revascularization. In this case, root canal treatment becomes compulsory and decreases the success of autotransplantation. The aim of this observational retrospective study was to evaluate whether partial resection of the root (performed during the autotransplantation of mature canines) would enable revascularization of the pulp after the surgery. METHODS: Five transmigrated mandibular canines with complete apex formation before the surgery were evaluated. During the autotransplantation surgery, the resection of 2 to 4 mm of the root apex was performed to open the path for revascularization of the pulp after surgery. The transplanted teeth were observed during healing after the surgery for the presence of pulp obliteration. Clinical and radiographic examinations were performed. RESULTS: All treated canines survived the minimum observation period of 2 years (ranging from 26 to 80 months, mean: 55 months) without pulp healing complications. The survival was 100%, and the success was 80%. In one canine, the external cervical root resorption was diagnosed and treated 1 year after the surgery. At the final examination, transplanted canines presented radiographic features of pulp obliteration, normal mobility, and healthy periodontal tissues. CONCLUSIONS: The surgical protocol proved to be successful in promoting revascularization to maintain pulp vitality, in all cases. The outcomes confirm that autotransplantation, combined with the resection of the root, constitutes a valid treatment for mature ectopic canines.

Design and synthesis of a new series of hybrids of functionalised N1-[(1H-1,2,3-triazol-4-yl)methyl]quinazoline-2,4-dione with antiviral activity against Respiratory Syncytial Virus.

In this study, a series of 48 hybrids of the functionalised 1-[(1H-1,2,3-triazole-4-yl)methyl]quinazoline-2,4-dione 17-22 were synthesised and evaluated for potential antiviral activity. The new hybrids were designed to contain a diethoxyphosphoryl group connected to the triazole moiety via ethylene or propylene linker, and in which the benzyl or benzoyl function is substituted at N3 in the quinazoline-2,4-dione moiety. The Cu(I)-catalyzed Hüisgen dipolar cycloaddition of azidophosphonates 23 and 24 with the respective N1-propargylquinazoline-2,4-diones 26aa-26ag, 26ba-26bg, 27aa-27ad and 27ba-27bd was applied for the syntheses of the designed compounds. All final hybrids 17-22 and N3-functionalised N1-propargylquinazoline-2,4-diones 26 and 27 were subsequently evaluated for their antiviral activity toward a broad range of DNA and RNA viruses. Importantly, hybrids 19be-19bg and 20be-20bg showed profound antiviral activities against Respiratory Syncytial Virus (RSV) with EC50 values in the lower micromolar range, with activity against viral strains of both subtypes (RSV A and B). In addition, several compounds also exerted some weak antiviral activity against varicella zoster virus. Finally, 19 ag was the only compound that showed antiviral potency against human cytomegalovirus, although with rather weak inhibitory activity. Notably, none of the tested compounds was cytotoxic toward uninfected cell lines used for the antiviral assays at a concentration up to 100 µM, returning interesting therapeutic indices for respiratory syncytial virus.

Role of Extracellular Matrix and Inflammation in Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is one of the most dangerous cardiovascular diseases, occurring mainly in men over the age of 55 years. As it is asymptomatic, patients are diagnosed very late, usually when they suffer pain in the abdominal cavity. The late detection of AAA contributes to the high mortality rate. Many environmental, genetic, and molecular factors contribute to the development and subsequent rupture of AAA. Inflammation, apoptosis of smooth muscle cells, and degradation of the extracellular matrix in the AAA wall are believed to be the major molecular processes underlying AAA formation. Until now, no pharmacological treatment has been implemented to prevent the formation of AAA or to cure the disease. Therefore, it is important that patients are diagnosed at a very early stage of the disease. Biomarkers contribute to the assessment of the concentration level, which will help to determine the level and rate of AAA development. The potential biomarkers today include homocysteine, cathepsins, osteopontin, and osteoprotegerin. In this review, we describe the major aspects of molecular processes that take place in the aortic wall during AAA formation. In addition, biomarkers, the monitoring of which will contribute to the prompt diagnosis of AAA patients over the age of 55 years, are described.

Rosemary Extract-Induced Autophagy and Decrease in Accumulation of Collagen Type I in Osteogenesis Imperfecta Skin Fibroblasts.

Osteogenesis imperfecta (OI) is a heterogeneous connective tissue disease mainly caused by structural mutations in type I collagen. Mutant collagen accumulates intracellularly, causing cellular stress that has recently been shown to be phenotype-related. Therefore, the aim of the study was to search for potential drugs reducing collagen accumulation and improving OI fibroblast homeostasis. We found that rosemary extract (RE), which is of great interest to researchers due to its high therapeutic potential, at concentrations of 50 and 100 µg/mL significantly reduced the level of accumulated collagen in the fibroblasts of four patients with severe and lethal OI. The decrease in collagen accumulation was associated with RE-induced autophagy as was evidenced by an increase in the LC3-II/LC3-I ratio, a decrease in p62, and co-localization of type I collagen with LC3-II and LAMP2A by confocal microscopy. The unfolded protein response, activated in three of the four tested cells, and the level of pro-apoptotic markers (Bax, CHOP and cleaved caspase 3) were attenuated by RE. In addition, the role of RE-modulated proteasome in the degradation of unfolded procollagen chains was investigated. This study provides new insight into the beneficial effects of RE that may have some implications in OI therapy targeting cellular stress.

Host and bacterial factors linking periodontitis and rheumatoid arthritis.

Observations from numerous clinical, epidemiological and serological studies link periodontitis with severity and progression of rheumatoid arthritis. The strong association is observed despite totally different aetiology of these two diseases, periodontitis being driven by dysbiotic microbial flora on the tooth surface below the gum line, while rheumatoid arthritis being the autoimmune disease powered by anti-citrullinated protein antibodies (ACPAs). Here we discuss genetic and environmental risk factors underlying development of both diseases with special emphasis on bacteria implicated in pathogenicity of periodontitis. Individual periodontal pathogens and their virulence factors are argued as potentially contributing to putative causative link between periodontal infection and initiation of a chain of events leading to breakdown of immunotolerance and development of ACPAs. In this respect peptidylarginine deiminase, an enzyme unique among prokaryotes for Porphyromonas gingivalis, is elaborated as a potential mechanistic link between this major periodontal pathogen and initiation of rheumatoid arthritis development.

Staphylococcal saoABC Operon Codes for a DNA-Binding Protein SaoC Implicated in the Response to Nutrient Deficit.

Whilst a large number of regulatory mechanisms for gene expression have been characterised to date, transcription regulation in bacteria still remains an open subject. In clinically relevant and opportunistic pathogens, such as Staphylococcus aureus, transcription regulation is of great importance for host-pathogen interactions. In our study we investigated an operon, exclusive to staphylococci, that we name saoABC. We showed that SaoC binds to a conserved sequence motif present upstream of the saoC gene, which likely provides a negative feedback loop. We have also demonstrated that S. aureus ΔsaoB and ΔsaoC mutants display altered growth dynamics in non-optimal media; ΔsaoC exhibits decreased intracellular survival in human dermal fibroblasts, whereas ΔsaoB produces an elevated number of persisters, which is also elicited by inducible production of SaoC in ΔsaoBΔsaoC double mutant. Moreover, we have observed changes in the expression of saoABC operon genes during either depletion of the preferential carbon or the amino acid source as well as during acidification. Comparative RNA-Seq of the wild type and ΔsaoC mutant demonstrated that SaoC influences transcription of genes involved in amino acid transport and metabolism, and notably of those coding for virulence factors. Our results suggest compellingly that saoABC operon codes for a DNA-binding protein SaoC, a novel staphylococcal transcription factor, and its antagonist SaoB. We linked SaoC to the response to nutrient deficiency, a stress that has a great impact on host-pathogen interactions. That impact manifests in SaoC influence on persister formation and survival during internalisation to host cells, as well as on the expression of genes of virulence factors that may potentially result in profound alternations in the pathogenic phenotype. Investigation of such novel regulatory mechanisms is crucial for our understanding of the dynamics of interactions between pathogenic bacteria and host cells, particularly in the case of clinically relevant, opportunistic pathogens such as Staphylococcus aureus.

Exclusive Enteral Nutrition Remission Induction Treatment Influence on Transient Hypertransaminasemia in Children with Newly Onset Crohn's Disease.

Pediatric Crohn's disease (CD) is commonly associated with hepatobiliary complications, including transient liver enzymes elevation (LEE). Exclusive enteral nutrition (EEN) is recommended as primary treatment in mild-to-moderate pediatric CD. Data concerning EEN and liver enzymes (LE) abnormalities are limited. The aim of this study was to assess the LEE occurrence in newly diagnosed CD pediatric patients during EEN. Retrospective analysis of 73 patients, with no previous signs of liver disease, qualified to EEN. LE were assessed at diagnosis, during EEN, after completion of the nutritional treatment, and reintroduction of free diet. Thirty-one (42%) children presented with LEE and 28 (38%) with transient LEE. The LEE cohort presented with higher percentage of protein energy (24.0% ± 29.4 IQR [interquartile range] vs. 18.6% ± 23.6 IQR, P < .05) versus nonprotein energy (fat and carbohydrates) in total energy intake (75.9% ± 29.4 IQR vs. 81.4% ± 23.6 IQR, P < .05). Also, the protein/energy ratio was higher in the LEE group compared with the group with normal LE (0.026 vs. 0.024, P = .028). At the fourth week of EEN, aspartate aminotransferase elevation correlated with higher daily protein intake (P < .018). The LEE during EEN is typically a low-grade and transient condition that may be connected to applied treatment. We hypothesize that higher protein/energy ratio during EEN may be associated with mild, temporary LEE. Careful observation with repeated measurement of LE activity may be sufficient proceeding in patients without any other symptoms of CD-associated liver disease.

Clinics and genetic background of hereditary gingival fibromatosis.

BACKGROUND: Hereditary gingival fibromatosis (HGF) is a rare condition characterized by slowly progressive overgrowth of the gingiva. The severity of overgrowth may differ from mild causing phonetic and masticatory issues, to severe resulting in diastemas or malposition of teeth. Both, autosomal-dominant and autosomal-recessive forms of HGF are described. The aim of this review is a clinical overview, as well as a summary and discussion of the involvement of candidate chromosomal regions, pathogenic variants of genes, and candidate genes in the pathogenesis of HGF. The loci related to non-syndromic HGF have been identified on chromosome 2 (GINGF, GINGF3), chromosome 5 (GINGF2), chromosome 11 (GINGF4), and 4 (GINGF5). Of these loci, pathogenic variants of the SOS-1 and REST genes inducing HGF have been identified in the GINGF and the GINGF5, respectively. Furthermore, among the top 10 clusters of genes ranked by enrichment score, ATP binding, and fibronectin encoding genes were proposed as related to HGF. CONCLUSION: The analysis of clinical reports as well as translational genetic studies published since the late'90s indicate the clinical and genetic heterogeneity of non-syndromic HGF and point out the importance of genetic studies and bioinformatics of more numerous unrelated families to identify novel pathogenic variants potentially inducing HGF. This strategy will help to unravel the molecular  mechanisms as well as uncover specific targets for novel and less invasive therapies of this rare, orphan condition.

Osteogenesis Imperfecta: Current and Prospective Therapies.

Osteogenesis Imperfecta (OI) is a group of connective tissue disorders with a broad range of phenotypes characterized primarily by bone fragility. The prevalence of OI ranges from about 1:15,000 to 1:20,000 births. Five types of the disease are commonly distinguished, ranging from a mild (type I) to a lethal one (type II). Types III and IV are severe forms allowing survival after the neonatal period, while type V is characterized by a mild to moderate phenotype with calcification of interosseous membranes. In most cases, there is a reduction in the production of normal type I collagen (col I) or the synthesis of abnormal collagen as a result of mutations in col I genes. Moreover, mutations in genes involved in col I synthesis and processing as well as in osteoblast differentiation have been reported. The currently available treatments try to prevent fractures, control symptoms and increase bone mass. Commonly used medications in OI treatment are bisphosphonates, Denosumab, synthetic parathyroid hormone and growth hormone for children therapy. The main disadvantages of these therapies are their relatively weak effectiveness, lack of effects in some patients or cytotoxic side effects. Experimental approaches, particularly those based on stem cell transplantation and genetic engineering, seem to be promising to improve the therapeutic effects of OI.

An Overview of Methods and Tools for Transfection of Eukaryotic Cells in vitro.

Transfection is a powerful analytical tool enabling studies of gene products and functions in eukaryotic cells. Successful delivery of genetic material into cells depends on DNA quantity and quality, incubation time and ratio of transfection reagent to DNA, the origin, type and the passage of transfected cells, and the presence or absence of serum in the cell culture. So far a number of transfection methods that use viruses, non-viral particles or physical factors as the nucleic acids carriers have been developed. Among non-viral carriers, the cationic polymers are proposed as the most attractive ones due to the possibility of their chemical structure modification, low toxicity and immunogenicity. In this review the delivery systems as well as physical, biological and chemical methods used for eukaryotic cells transfection are described and discussed.

The Stimulating Effect of Rosmarinic Acid and Extracts from Rosemary and Lemon Balm on Collagen Type I Biosynthesis in Osteogenesis Imperfecta Type I Skin Fibroblasts.

Rosemary extract (RE) and lemon balm extract (LBE) attract particular attention of pharmacists due to their high therapeutic potential. Osteogenesis imperfecta (OI) type I is a heritable disease caused by mutations in type I collagen and characterized by its reduced amount. The aim of the study was to evaluate the effect of the extracts and rosmarinic acid (RA) on collagen type I level in OI skin fibroblasts. Phytochemical analysis of RE and LBE was carried out by liquid chromatography-photodiode array detection-mass spectrometry. The expression of collagen type I at transcript and protein levels was analyzed by qPCR, ELISA, SDS-urea PAGE, and Western blot. In OI patient's fibroblasts the exposure to the extracts (0.1-100 µg/mL) and RA (0.1-100 µM) significantly increased collagen type I and the best results were obtained with 0.1-10 µM RA and 0.1-10 µg/mL of the extracts. LBE showed a greater stimulating effect than RE, likely due to a higher RA content. Moreover, collagen type III expression and matrix metalloproteinase (MMP-1, -2, -9) activity remained unchanged or decreased. The obtained data support the clinical potential of RA-rich extracts and RA itself in modulating the quantitative defect of type I collagen in type I OI.

Periodontal condition of mandibular incisors treated with modified Kazanjian vestibuloplasty compared to untreated sites: A prospective study.

BACKGROUND: A shallow vestibule, insufficient keratinized tissue width and pulling of marginal gingiva may be associated with gingival recession, plaque accumulation and gingivitis. Conventional techniques for treatment of gingival recession use autogenous or allogenic grafts. However, these methods result in soreness at the donor site and pose an economic burden, which may cause patients to withdraw from treatment. Alternative therapy is currently not available to treat such patients. OBJECTIVES: The aim of this study was to evaluate changes in periodontal tissue at the mandibular incisors after vestibuloplasty, focusing on functional improvement of the existing soft tissue with no grafting. MATERIAL AND METHODS: Thirty patients with a shallow vestibule, minimal keratinized tissue width (KTW; ≤1 mm), gingival recession (REC) and pulling of gingiva underwent modified Kazanjian vestibuloplasty were included into the test group, whereas 27 patients did not undergo any surgery (control group). The probing pocket depth (PPD), clinical attachment level (CAL), gingival recession depth (GRD), and KTW were assessed at baseline and 12 months post-surgery. RESULTS: The mean KTW, GRD and CAL values improved in the test group. A significant increase in mean KTW value (1.17 ±1.22 mm, p = 0.0406) was detected in the test group, while the control group showed a further reduction in mean KTW value (0.13 ±0.45 mm). The mean GRD value decreased from 2.09 ±1.78 mm to 1.22 ±1.46 mm (p = 0.0087) in the test group, whereas in controls the mean GRD value increased from 1.95 ±1.29 mm to 2.34 ±1.44 mm (p = 0.0164). The mean KTW value at 3, 6 and 12 months compared to baseline showed an increase in the test group, and the mean GRD and CAL values exhibited the potential to improve. CONCLUSIONS: Sites treated with vestibuloplasty showed increased KTW, improvement in the gingival margin and CAL gain, whereas untreated sites showed continuous deterioration of the evaluated parameters. Vestibuloplasty may be recommended for patients avoiding major surgery for which functional improvement in tissue alone would provide a sufficient therapeutic outcome.

Antimicrobial photodynamic therapy effectively reduces Porphyromonas gingivalis infection in gingival fibroblasts and keratinocytes: An in vitro study.

BACKGROUND: Porphyromonas gingivalis possess the ability to invade host cells which prevents this pathogen from eradication by conventional periodontal therapy. Recently, antimicrobial photodynamic therapy (aPDT) was introduced to periodontal treatment as a complementary antibacterial method. The aim of this study was to evaluate the effect of toluidine blue-O (TBO) mediated aPDT on the viability of P. gingivalis invading gingival fibroblasts and keratinocytes in an in vitro model of infection. METHODS: Primary human gingival fibroblasts (PHGF) and telomerase immortalized gingival keratinocytes (TIGK) were infected with Pg ATCC 33277. Two concentrations of TBO (0.01 mg/mL, TBO-c1 and 0.001 mg/mL, TBO-c2) and a non-laser red light source (λ = 630 nm) were applied to treat both cell-adherent/intracellular Pg (the adhesion/invasion model) or exclusively the intracellular bacteria (the intracellular infection model). RESULTS: The median viability of cell-adherent/intracellular Pg in infected keratinocytes declined from 1.88 × 105 cfu/mL in infected cells treated with TBO without irradiation to 40 cfu/mL upon irradiation for 10 s with TBO-c1. At higher light doses a complete photokilling of P. gingivalis was observed. Pg from exclusively intracellular infection model was also efficiently eradicated as the residual viability dropped from 1.44 × 105 cfu/mL in control samples to 160, 20 and 10 cfu/mL upon irradiation for 10, 20 and 30 s, respectively. In the infected fibroblasts irradiation significantly reduced bacterial viability but did not completely eradicate the intracellular pathogen. CONCLUSIONS: Antimicrobial PDT is effective in reducing the viability of intracellular periopathogens, however those residing within gingival fibroblasts seems to attenuate the photokilling effectiveness of this method.

Circulating miRNA-181b-5p, miRNA-223-3p, miRNA-210-3p, let 7i-5p, miRNA-21-5p and miRNA-29a-3p in patients with localized scleroderma as potential biomarkers.

Localized scleroderma (LoSc) is a rare disease manifested by an inflammation and sclerosis of the skin. The latest studies focused on glycoprotein Krebs von den Lungen-6, surfactant protein-D, chemokine ligand 18 and dipeptidylpeptidase 4 as potential biomarkers of skin fibrosis in systemic scleroderma. Our study aimed to identify 6 miRNAs with elevated or decreased levels in 38 LoSc patients (31 females, 7 males) compared to healthy volunteers (HVs) and to correlate the selected miRNAs' serum levels with the severity and the clinical symptoms of LoSc and some laboratory parameters with the selected miRNAs' serum levels. The serum levels of miRNAs, i.e. miRNA-181b-5p, miRNA-223-3p, miRNA-21-5p, let 7i-5p, miRNA-29a-3p and miRNA-210-3p were significantly increased in the LoSc patients compared to the HVs. The level of let-7i increase in the female LoSc patients correlated negatively with BSA (r = - 0.355, p = 0.049) and mLoSSI (r = - 0.432, p = 0.015). Moreover, the female patients with inactive LoSc had significantly higher level of let-7i (2.68-fold on average) in comparison to those with active disease (p = 0.045). The exact role of those molecules has not been revealed in LoSc and a long-term longitudinal research is pivotal to confirm their prognostic value.

Candida albicans Shields the Periodontal Killer Porphyromonas gingivalis from Recognition by the Host Immune System and Supports the Bacterial Infection of Gingival Tissue.

Candida albicans is a pathogenic fungus capable of switching its morphology between yeast-like cells and filamentous hyphae and can associate with bacteria to form mixed biofilms resistant to antibiotics. In these structures, the fungal milieu can play a protective function for bacteria as has recently been reported for C. albicans and a periodontal pathogen-Porphyromonas gingivalis. Our current study aimed to determine how this type of mutual microbe protection within the mixed biofilm affects the contacting host cells. To analyze C. albicans and P. gingivalis persistence and host infection, several models for host-biofilm interactions were developed, including microbial exposure to a representative monocyte cell line (THP1) and gingival fibroblasts isolated from periodontitis patients. For in vivo experiments, a mouse subcutaneous chamber model was utilized. The persistence of P. gingivalis cells was observed within mixed biofilm with C. albicans. This microbial co-existence influenced host immunity by attenuating macrophage and fibroblast responses. Cytokine and chemokine production decreased compared to pure bacterial infection. The fibroblasts isolated from patients with severe periodontitis were less susceptible to fungal colonization, indicating a modulation of the host environment by the dominating bacterial infection. The results obtained for the mouse model in which a sequential infection was initiated by the fungus showed that this host colonization induced a milder inflammation, leading to a significant reduction in mouse mortality. Moreover, high bacterial counts in animal organisms were noted on a longer time scale in the presence of C. albicans, suggesting the chronic nature of the dual-species infection.