Comparison of paperpoint and curette sampling of subgingival microbiome composition as analyzed by 16S rRNA gene amplicon sequencing

Objective: Our aim was to compare the subgingival microbiome composition in samples collected by curettes and paperpoints. Methods: Subgingival plaque of rheumatoid arthritis patients with periodontitis (N=66) or gingivitis (N=15) was collected at two timepoints by sterile curettes and paperpoints. Unused sterile paperpoints were included as controls. The microbial DNA was processed for 16S rRNA gene amplicon sequencing. The data was processed and clustered into Operational Taxonomic Units (OTUs) and assessed by multivariate analyses. Results: Unused paperpoints showed various levels of DNA contamination. The OTUs specific for unused paperpoints were classified as Exiguobacterium, Enterococcus, Methylobacterium, Aquabacterium and Pseudomonas and were removed from the dataset. Microbial profiles of curette samples differed significantly from the paperpoint samples (PERMANOVA, p=0.0009, F=3.167). The paperpoint samples had significantly higher proportion of OTUs classified as Streptococcus, Gemella, Parvimonas, Haemophilus, Aggregatibacter and Clostridiales family XIII incertae sedis (p<0.05). Curette samples harbored higher proportion of Corynebacterium, Prevotella, Selenomonas, Actinomyces and Treponema (p<0.05). Curette samples had significantly higher species richness (p=0.01) and Shannon Diversity Index (p=0.009) than the paperpoint samples. Conclusions: Different subgingival plaque sampling techniques result in different microbiome profiles. Samples by paperpoints introduce microbial DNA contaminants and show underestimated microbial diversity compared to samples obtained by curettes

A role for alpha 11 beta 1 integrin in the human periodontal ligament

We previously demonstrated a role for α11β1 integrin in periodontal ligament (PDL)-driven tooth eruption in the mouse. To explore a possible role for α11β1 in the human periodontium, we have characterized the expression and function of α11 in human PDL tissue, in human PDL fibroblasts (hPDLF), and in human gingival fibroblasts (hGF). α11 expression was detected in PDL tissue, in hPDLF, and in hGF cells. Platelet-derived growth factor-BB and insulin-like growth factor II stimulated contraction of collagen lattices by both types of fibroblasts. α2 integrin blocking antibodies and the use of α11 siRNA demonstrated a role for both α2β1 and α11β1 in collagen lattice remodeling. Analysis of the proximal ITGA11 promoter from persons with chronic periodontal disease failed to reveal any polymorphism. Analysis of our data shows that α11β1 is a major collagen receptor on cultured human PDL cells and implies that it is also functionally important in the PDL in vivo

Lymphotoxin-beta receptor blockade reduces CXCL13 in lacrimal glands and improves corneal integrity in the NOD model of Sjögren&apos;s syndrome

Introduction: In Sjögren&apos;s syndrome, keratoconjunctivitis sicca (dry eye) is associated with infiltration of lacrimal glands by leukocytes and consequent losses of tear-fluid production and the integrity of the ocular surface. We investigated the effect of blockade of the lymphotoxin-beta receptor (LTBR) pathway on lacrimal-gland pathology in the NOD mouse model of Sjögren&apos;s syndrome. Methods: Male NOD mice were treated for up to ten weeks with an antagonist, LTBR-Ig, or control mouse antibody MOPC-21. Extra-orbital lacrimal glands were analyzed by immunohistochemistry for high endothelial venules (HEV), by Affymetrix gene-array analysis and real-time PCR for differential gene expression, and by ELISA for CXCL13 protein. Leukocytes from lacrimal glands were analyzed by flow-cytometry. Tear-fluid secretion-rates were measured and the integrity of the ocular surface was scored using slit-lamp microscopy and fluorescein isothiocyanate (FITC) staining. The chemokine CXCL13 was measured by ELISA in sera from Sjögren&apos;s syndrome patients (n = 27) and healthy controls (n = 30). Statistical analysis was by the two-tailed, unpaired T-test, or the Mann-Whitney-test for ocular integrity scores. Results: LTBR blockade for eight weeks reduced B-cell accumulation (approximately 5-fold), eliminated HEV in lacrimal glands, and reduced the entry rate of lymphocytes into lacrimal glands. Affymetrix-chip analysis revealed numerous changes in mRNA expression due to LTBR blockade, including reduction of homeostatic chemokine expression. The reduction of CXCL13, CCL21, CCL19 mRNA and the HEV-associated gene GLYCAM-1 was confirmed by PCR analysis. CXCL13 protein increased with disease progression in lacrimal-gland homogenates, but after LTBR blockade for 8 weeks, CXCL13 was reduced approximately 6-fold to 8.4 pg/mg (+/- 2.7) from 51 pg/mg (+/-5.3) in lacrimal glands of 16 week old control mice. Mice given LTBR blockade exhibited an approximately two-fold greater tear-fluid secretion than control mice (P = 0.001), and had a significantly improved ocular surface integrity score (P = 0.005). The mean CXCL13 concentration in sera from Sjögren&apos;s patients (n = 27) was 170 pg/ml, compared to 92.0 pg/ml for sera from (n = 30) healthy controls (P = 0.01). Conclusions: Blockade of LTBR pathways may have therapeutic potential for treatment of Sjögren&apos;s syndrome. © 2011 Fava et al

Genetic diversity of oral Fusobacterium nucleatum isolated from patients with different clinical conditions Diversidade genética de Fusobacterium nucleatum orais isolados de pacientes com diferentes condições clínicas

The genetic diversity of 23 oral Fusobacterium nucleatum isolated from 15 periodontal patients, eight from seven healthy subjects, nine from nine AIDS patients and two from two Cebus apella monkeys were analyzed. EcoRI restricted the bacterial DNA and 28 ribotypes grouped from A to J groups were obtained. Isolates formed 24 ribotypes which were contained into A, B, C, D, E and F groups, and three reference strains and two clinical isolates of A. actinomycetemcomitans, and E. coli CDC formed four different ribotypes into the G, H, I and J groups. Moreover, from nine F. nucleatum from AIDS patients, six were ribotyped as group C and three as group D. By using ribotyping we distinguished F. nucleatum recovered from different sources. It is possible that isolates from AIDS patients may contain some phenotypic or genotypic factor did not observed in this study.<br>Neste estudo foi avaliada a diversidade genética de 23 amostras de Fusobacterium nucleatum isoladas da cavidade bucal de 15 pacientes com doença periodontal, de oito cepas isoladas de sete indivíduos sadios, de nove isoladas de nove pacientes com AIDS e de duas isoladas de dois macacos Cebus apella. Pela ação da enzima EcoRI sobre o DNA bacteriano foram reconhecidos 28 ribotipos agrupados de A a J. Os isolados testados formaram 24 ribotipos os quais foram contidos nos grupos A, B, C, D, E e F, e as três cepas de referência e dois isolados clínicos de A. actinomycetemcomitans e E. coli CDC formaram quatro diferentes ribotipos contidos nos grupos G, H, I e J. Em adição, as nove cepas de F. nucleatum isoladas de pacientes com AIDS, seis pertenciam ao grupo C e três ao grupo D. Usando-se a ribotipagem foi possível distinguir F. nucleatum isolados de diferentes origens