Katarzyna Gawron

Porphyromonas gingivalis Facilitates the Development and Progression of Destructive Arthritis through Its Unique Bacterial Peptidylarginine Deiminase (PAD)

Rheumatoid arthritis and periodontitis are two prevalent chronic inflammatory diseases in humans and are associated with each other both clinically and epidemiologically. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic autoimmune response to citrullinated epitopes. Here we showed that infection with viable periodontal pathogen Porphyromonas gingivalis strain W83 exacerbated collagen-induced arthritis (CIA) in a mouse model, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique P. gingivalis peptidylarginine deiminase (PPAD), which converts arginine residues in proteins to citrulline. Infection with wild type P. gingivalis was responsible for significantly increased levels of autoantibodies to collagen type II and citrullinated epitopes as a PPAD-null mutant did not elicit similar host response. High level of citrullinated proteins was also detected at the site of infection with wild-type P. gingivalis. Together, these results suggest bacterial PAD as the mechanistic link between P. gingivalis periodontal infection and rheumatoid arthritis.

Gingival fibromatosis: clinical, molecular and therapeutic issues

Gingival fibromatosis is a rare and heterogeneous group of disorders that develop as slowly progressive, local or diffuse enlargements within marginal and attached gingiva or interdental papilla. In severe cases, the excess tissue may cover the crowns of the teeth, thus causing functional, esthetic, and periodontal problems, such as bone loss and bleeding, due to the presence of pseudopockets and plaque accumulation. It affects both genders equally. Hereditary, drug-induced, and idiopathic gingival overgrowth have been reported. Hereditary gingival fibromatosis can occur as an isolated condition or as part of a genetic syndrome. The pathologic manifestation of gingival fibromatosis comprises excessive accumulation of extracellular matrix proteins, of which collagen type I is the most prominent example. Mutation in the Son-of-Sevenless-1 gene has been suggested as one possible etiological cause of isolated (non-syndromic) hereditary gingival fibromatosis, but mutations in other genes are also likely to be involved, given the heterogeneity of this condition. The most attractive concept of mechanism for drug-induced gingival overgrowth is epithelial-to-mesenchymal transition, a process in which interactions between gingival cells and the extracellular matrix are weakened as epithelial cells transdifferentiate into fibrogenic fibroblast-like cells. The diagnosis is mainly made on the basis of the patient’s history and clinical features, and on histopathological evaluation of affected gingiva. Early diagnosis is important, mostly to exclude oral malignancy. Differential diagnosis comprises all pathologies in the mouth with excessive gingival overgrowth. Hereditary gingival fibromatosis may present as an autosomal-dominant or less commonly autosomal-recessive mode of inheritance. If a systemic disease or syndrome is suspected, the patient is directed to a geneticist for additional clinical examination and specialized diagnostic tests. Treatments vary according to the type of overgrowth and the extent of disease progression, thus, scaling of teeth is sufficient in mild cases, while in severe cases surgical intervention is required. Prognosis is precarious and the risk of recurrence exists.

Gingipains: Critical Factors in the Development of Aspiration Pneumonia Caused by Porphyromonas gingivalis

Aspiration pneumonia is a life-threatening infectious disease often caused by oral anaerobic and periodontal pathogens such as Porphyromonas gingivalis. This organism produces proteolytic enzymes, known as gingipains, which manipulate innate immune responses and promote chronic inflammation. Here, we challenged mice with P. gingivalis W83 and examined the role of gingipains in bronchopneumonia, lung abscess formation, and inflammatory responses. Although gingipains were not required for P. gingivalis colonization and survival in the lungs, they were essential for manifestation of clinical symptoms and infection-related mortality. Pathologies caused by wild-type (WT) P. gingivalis W83, including hemorrhage, necrosis, and neutrophil infiltration, were absent from lungs infected with gingipain-null isogenic strains or WT bacteria preincubated with gingipain-specific inhibitors. Damage to lung tissue correlated with systemic inflammatory responses, as manifested by elevated levels of TNF, IL-6, IL-17, and C-reactive protein. These effects were unequivocally dependent on gingipain activity. Gingipain activity was also implicated in the observed increase in IL-17 in lung tissues. Furthermore, gingipains increased platelet counts in the blood and activated platelets in the lungs. Arginine-specific gingipains made a greater contribution to P. gingivalis-related morbidity and mortality than lysine-specific gingipains. Thus, inhibition of gingipain may be a useful adjunct treatment for P. gingivalis-mediated aspiration pneumonia.

Use of Sensitive, Broad-Spectrum Molecular Assays and Human Airway Epithelium Cultures for Detection of Respiratory Pathogens

Rapid and accurate detection and identification of viruses causing respiratory tract infections is important for patient care and disease control. Despite the fact that several assays are available, identification of an etiological agent is not possible in ∼30% of patients suffering from respiratory tract diseases. Therefore, the aim of the current study was to develop a diagnostic set for the detection of respiratory viruses with sensitivity as low as 1–10 copies per reaction. Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ∼76% of cases. To improve assay performance and facilitate the identification of novel species or emerging strains, cultures of fully differentiated human airway epithelium were used to pre-amplify infectious viruses. This additional step resulted in the detection of pathogens in all samples tested. Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used.

Persistence of intracellular and extracellular changes after incompletely suppressing expression of the R789C (p.R989C) and R992C (p.R1192C) collagen II mutants

Mutations in COL2A1 produce a spectrum of disorders whose hallmark feature is alterations in skeletal development. Attempts to counteract the effects of collagen mutations at the molecular level have been relatively ineffective due to the inability to selectively suppress a mutant allele, and failure to deliver a sufficient number of cells expressing wild‐type collagen. Moreover, these approaches are hampered because the minimal therapeutic conditions that would allow extracellular matrix remodeling and recovery of cells from stress are not known. Here, we employed a tetracycline‐inducible system for expressing the R789C or R992C collagen II mutants, allowing us to decrease the production of mutant proteins by 25, 50, 75, or 100% with respect to their initial production. Through analysis of intracellular and extracellular parameters we have shown that affected cell/matrix systems are able to recover from mutation‐induced aberrations only when 100% expression of mutant collagens is shut off, but not if the expression of small amounts of mutant molecules persists in the system. Our data suggest that efficient remodeling of tissues affected by the presence of thermolabile collagen mutants may depend on their complete elimination rather than on partial reduction. Hum Mutat 32:1–12, 2011.

Reducing the effects of intracellular accumulation of thermolabile collagen II mutants by increasing their thermostability in cell culture conditions

Mutations in collagen II are associated with spondyloepiphyseal dysplasia, a group of heritable diseases whose common features include aberrations of skeletal growth. The mechanisms through which mutations in collagen II affect the cartilaginous tissues are complex and include both intracellular and extracellular processes. One of those mechanisms involves cellular stress caused by excessive accumulation of misfolded collagen II mutants. We investigated whether stabilizing the structure of thermolabile R789C and R992C collagen II mutants would improve their secretion from cells, thereby reducing cellular stress and apoptosis. Employing glycerol and trimethylamine N-oxide (TMAO), chemicals that increase the thermostability of collagen triple helices, we demonstrated that those compounds function as chaperones and stabilize the R789C and R992C mutants, accelerate their secretion, and improve cell survival. Our study provides a scientific basis for considering misfolded triple helices of collagen mutants a target for reducing the deleterious effects caused by their excessive intracellular accumulation.

Gingival Fibromatosis with Significant De Novo Formation of Fibrotic Tissue and a High Rate of Recurrence.

Patient: Female, 11 Final Diagnosis: Hereditary gingival fibromatosis Symptoms: Gingival overgrowth Medication: — Clinical Procedure: Surgery Specialty: Dentistry Objective: Rare disease Background: Hereditary gingival fibromatosis is characterized by slowly progressive enlargement of the gingiva that can present as an isolated condition or a part of various syndromes. Case Report: An 11-year-old female reported with a gingival lesion that caused masticatory problems and poor oral hygiene. Periodontal examination revealed a dense tissue covering 30% of her teeth crowns within both jaws. Panoramic x-ray showed a normal bone height and teeth positioning. The patient did not use any medications, but a similar condition was also present in other family members. The patient was diagnosed with hereditary gingival fibromatosis. Surgery was carried out to remove excess of gingival tissue. Post-surgical healing was uneventful, but four weeks after the first surgery, the condition recurred amounting to 45% of the initial tissue volume presenting in the mandible, and 25% in the maxilla. Two months later, no significant growth was noted in the mandible, while in the maxilla, growth increased to 40% of the pre-operative state. Analysis by polarized microscope showed a significant increase of thin fibrotic fibrils that contributed 80% of the total pool of collagen fibrils in the patient’s gingiva, but only 25% in healthy gingiva. The patient was receiving outpatient care for follow-up every three months and surgical intervention had not been planned as long as her periodontal health would not be compromised. Conclusions: It is currently not clear whether the extent of the fibrosis had a mechanistic association with the ratio of gingival tissue re-growth in our case study. Further studies are needed to explain this association and improve the management of this condition.

Analysis of mutations in the SOS-1 gene in two Polish families with hereditary gingival fibromatosis

OBJECTIVES To establish whether two families from Malopolska and Mazovia provinces in Poland are affected by hereditary gingival fibromatosis type 1, caused by a single-cytosine insertion in exon 21 of the Son-of-Sevenless-1 gene. MATERIAL AND METHODS Six subjects with hereditary gingival fibromatosis and five healthy subjects were enrolled in the study. Gingival biopsies were collected during gingivectomy or tooth extraction and used for histopathological evaluation. Total RNA and genomic DNA were purified from cultured gingival fibroblasts followed by cDNA and genomic DNA sequencing and analysis. RESULTS Hereditary gingival fibromatosis was confirmed by periodontal examination, X-ray, and laboratory tests. Histopathological evaluation showed hyperplastic epithelium, numerous collagen bundles, and abundant-to-moderate fibroblasts in subepithelial and connective tissue. Sequencing of exons 19-22 of the Son-of-Sevenless-1 gene did not reveal a single-cytosine insertion nor other mutations. CONCLUSIONS Patients from two Polish families under study had not been affected by hereditary gingival fibromatosis type 1, caused by a single-cytosine insertion in exon 21 of the Son-of-Sevenless-1 gene. Further studies of the remaining regions of this gene as well as of other genes are needed to identify disease-related mutations in these patients. This will help to unravel the pathogenic mechanism of gingival overgrowth.

TIMP-1 association with collagen type I overproduction in hereditary gingival fibromatosis

OBJECTIVES To investigate the processes associated with the excessive production of collagen I in hereditary gingival fibromatosis (HGF). MATERIALS AND METHODS Three HGF subjects and five controls were enrolled in the study. Histomorphological and immunohistological analyses were performed on gingival tissues. The expression of heat-shock protein 47 (HSP47), collagen I, transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) by gingival fibroblasts isolated from HGF and controls was analysed using qRT-PCR, Western blotting and ELISA. RESULTS Considerable accumulation of fibrotic fibrils and increased synthesis of HSP47 were noted in HGF gingival tissues. The synthesis of collagen I, HSP47, TGF-β1, CTGF and TIMP-1 was significantly elevated in HGF gingival fibroblasts compared with controls, while the production of MMP-1 was decreased. CONCLUSIONS We report that fibrosis in HGF gingival tissues is associated with increased synthesis of HSP47. This finding was confirmed by an in vitro study, where excessive production of collagen I was associated with increased synthesis of HSP47, TGF-β1 and CTGF by HGF gingival fibroblasts. Moreover, the shift in the TIMP-1/MMP-1 ratio identifies increased synthesis of TIMP-1 as one of the processes associated with collagen I overproduction in HGF fibroblasts.

Endoplasmic reticulum stress in chondrodysplasias caused by mutations in collagen types II and X

The endoplasmic reticulum is primarily recognized as the site of synthesis and folding of secreted, membrane-bound, and some organelle-targeted proteins. An imbalance between the load of unfolded proteins and the processing capacity in endoplasmic reticulum leads to the accumulation of unfolded or misfolded proteins and endoplasmic reticulum stress, which is a hallmark of a number of storage diseases, including neurodegenerative diseases, a number of metabolic diseases, and cancer. Moreover, its contribution as a novel mechanistic paradigm in genetic skeletal diseases associated with abnormalities of the growth plates and dwarfism is considered. In this review, I discuss the mechanistic significance of endoplasmic reticulum stress, abnormal folding, and intracellular retention of mutant collagen types II and X in certain variants of skeletal chondrodysplasia.