Anne Karin Kristoffersen

Bacteria and bacterial DNA in atherosclerotic plaque and aneurysmal wall biopsies from patients with and without periodontitis

Background Several studies have reported an association between chronic periodontitis (CP) and cardiovascular diseases. Detection of periodontopathogens, including red complex bacteria (RCB), in vascular lesions has suggested these bacteria to be involved in the pathogenesis of atherosclerosis and abdominal aortic aneurysms. Objective In this study, we investigate bacteria and their DNA in vascular biopsies from patients with vascular diseases (VD; i.e. abdominal aortic aneurysms, atherosclerotic carotid, and common femoral arteries), with and without CP. Methods DNA was extracted from vascular biopsies selected from 40 VD patients: 30 with CP and 10 without CP. The V3-V5 region of the 16S rDNA (V3-V5) was polymerase chain reaction (PCR)-amplified, and the amplicons were cloned into Escherichia coli, sequenced, and classified (GenBank and the Human Oral Microbiome database). Species-specific primers were used for the detection of Porphyromonas gingivalis. In addition, 10 randomly selected vascular biopsies from the CP group were subjected to scanning electron microscopy (SEM) for visualization of bacteria. Checkerboard DNA–DNA hybridization was performed to assess the presence of RCB in 10 randomly selected subgingival plaque samples from CP patients. Results A higher load and mean diversity of bacteria were detected in vascular biopsies from VD patients with CP compared to those without CP. Enterobacteriaceae were frequently detected in vascular biopsies together with cultivable, commensal oral, and not-yet-cultured bacterial species. While 70% of the subgingival plaque samples from CP patients showed presence of RCB, only P. gingivalis was detected in one vascular biopsy. Bacterial cells were seen in all 10 vascular biopsies examined by SEM. Conclusions A higher bacterial load and more diverse colonization were detected in VD lesions of CP patients as compared to patients without CP. This indicated that a multitude of bacterial species both from the gut and the oral cavity, rather than exclusively periodontopathogens, may be involved as additional risk factors in the pathogenesis of VD.

The human polyomavirus BK T antigen induces gene expression in human cytomegalovirus.

Co-infections or co-habitations of cells by two or more viruses may occur in the human organism. Human cytomegalovirus (HCMV) and the human polyomavirus BK (BKV) have common host cells and may both establish lifelong latency/persistence following primary infection. Both viruses may become reactivated by immunosuppression or other conditions which upset host-virus balance, and they encode gene products with the inherent potential of acting as heterologous transacting factors for expression of cellular or viral genes. It has been shown that HCMV induces gene expression and replication of primate polyomaviruses. We now demonstrate that BKV is able to enhance the expression of HCMV immediate early (IE1 and 2) as well as the early (E) protein pp65 during double infections in semi-permissive cells. By transfection experiments it was established that the phenomenon is due to heterologous transcriptional transactivation of the HCMV major IE promoter (MIEP) by the BKV large T antigen, without contribution from the small t antigen.

Human papillomavirus subtypes in oral lesions compared to healthy oral mucosa

BACKGROUND Human papillomaviruses (HPV) are involved in the etiology of cervix cancer, but it is still unclear whether they play a role in related oral lesions. OBJECTIVES The presence of HPV in oral leukoplakia biopsies (n=50) and oral squamous carcinoma biopsies (n=50) was compared to normal oral mucosa swabs (n=50) for the purpose of indicating a possible etiological role for the virus. STUDY DESIGN DNA was extracted from tissue biopsies and from mucosa swabs of control samples. Nested PCR was performed with primers targeting conserved sequences within the capsid gene L1. PCR products were sequenced to identify the HPV genotype. RESULT The results reveal a profile of low-risk HPV genotypes in oral leukoplakia similar to that in healthy controls, while HPV was less frequently observed in oral squamous carcinoma. CONCLUSIONS HPV does not seem to represent an important causal factor for the development of oral leukoplakia or oral squamous carcinoma.

Simian Virus 40 Large T-antigen, but not Small T-antigen, Trans-activatesthe Human Cytomegalovirus Major Immediate Early Promoter

Cytomegalovirus infection is a major cause of morbidity in immunocompromised patients. The major immediate early promoter/enhancer (MIEP) of the human cytomegalovirus controls the expression of the immediate early genes 1 and 2 which play a central role both in primary and reactivated human cytomegalovirus (HCMV)-infections. Our previous studies have shown that co-infection of A549 cells with human cytomegalovirus and human polyomavirus BK resulted in enhanced expression of the immediate early genes 1 and 2 and that the early gene products of BK virus trans-activated the MIEP. However, neither the MIEP sequences required for mediating this trans-activation, nor the contribution of the individual BK virus early gene products were examined. The MIEP contains multiple binding sites for the transcription factors CREB, AP1, Sp1 and NFκB, which may mediate polyomavirus large T- or small t-antigens-induced promoter activation. Transient transfection studies in A549 cells demonstrated that SV40 large T-antigen, but not small t-antigen, trans-activated MIEP activity approximately 9-fold. Mutations in individual binding motifs in the context of the complete MIEP did not impair trans-activation by large T-antigen. The level of induction of a truncated MIEP consisting of a single set of CRE/AP1, NFκB, and Sp1 binding motifs by large T-antigen was reduced 2-fold compared to the full length MIEP. Extended truncations diminished trans-activation by large T-antigen. To determine the contribution of a single binding motif in the trans-activation by large T-antigen, a CRE/AP1, an NFκB, an Sp1, or a non-consensus Sp1-motif, respectively, was linked to the MIEP TATA-sequence respecting the natural spacing between the two transcription regulatory elements. Only the MIEP TATA-box with the correctly spaced non-consensus Sp1 binding site (GT-motif) was stimulated by large T-antigen. These results suggest that an isolated non-consensus Sp1-motif is important for trans-activation of the MIEP by large T-antigen, but that other cis-acting elements can compensate for this element in the context of the whole MIEP.

Interaction of herpes simplex virus with mononuclear phagocytes is dependent on the differentiation stage of the cells.

The interaction of herpes simplex virus (HSV) with mononuclear phagocytes (MP), i.e. monocytes and macrophages, is of importance for the pathogenesis of HSV infections. MP are known to play a significant role in the cellular defence against infections with HSV, but it has also been shown that HSV‐1 affects MP. The infection of these cells at different stages of differentiation has different outcomes, and may result in the alteration of important cellular functions. HSV‐1 inhibits the morphological differentiation of human monocytes, and this inhibition occurs in spite of the fact that human monocytes are non‐permissive to HSV‐1. We have studied the effect of HSV infection of monocytes and macrophages on production of essential cytokines and related this effect to the reproduction of the virus. Blood‐derived MP were cultured in vitro and inoculated with HSV at different stages of differentiation. Replication of the virus was measured by infectivity titration, detection of HSV antigens by immunofluorescence and detection of HSV‐specific mRNA. In monocytes, no viral replication and no production of late protein was seen. HSV IE gene was transcribed in monocytes from some donors, but not from others. In macrophages, virus replicated, but less efficiently than in fully permissive fibroblast cells. The production of IL‐1β, IL‐6 and TNF‐α in both non‐permissive monocytes and permissive macrophages was assayed both at the transcriptional level, as mRNA, and as protein released from the cells. Production of cytokines by MP was affected by HSV‐1. The level of cytokine mRNA and cytokine protein did not correspond for all cytokines, which may suggest that translational regulation and/or cytokine inhibitors are important in the regulation of the cytokine response. The cytokine modulation, both at the transcriptional level and measured as biological activity, was different in monocytes and macrophages, and varied between different donors. Our results indicate a relation between permissiveness and cytokine response in mononuclear phagocytes infected with HSV‐1. Such a relation may be of importance to both intrinsic and extrinsic defence mechanisms of MP against HSV‐1. Our study also demonstrates that even the functions of non‐permissive cells such as blood‐derived monocytes may be affected by viral infections.

Influence of the Apical Preparation Size and the Irrigant Type on Bacterial Reduction in Root Canal–treated Teeth with Apical Periodontitis

Introduction: This clinical study evaluated the influence of the apical preparation size using nickel‐titanium rotary instrumentation and the effect of a disinfectant on bacterial reduction in root canal–treated teeth with apical periodontitis. Methods: Forty‐three teeth with posttreatment apical periodontitis were selected for retreatment. Teeth were randomly divided into 2 groups according to the irrigant used (2.5% sodium hypochlorite [NaOCl], n = 22; saline, n = 21). Canals were prepared with the Twisted File Adaptive (TFA) system (SybronEndo, Orange, CA). Bacteriological samples were taken before preparation (S1), after using the first instrument (S2), and then after the third instrument of the TFA system (S3). In the saline group, an additional sample was taken after final irrigation with 1% NaOCl (S4). DNA was extracted from the clinical samples and subjected to quantitative real‐time polymerase chain reaction to evaluate the levels of total bacteria and streptococci. Results: S1 from all teeth were positive for bacteria. Preparation to the first and third instruments from the TFA system showed a highly significant intracanal bacterial reduction regardless of the irrigant (P < .01). Apical enlargement to the third instrument caused a significantly higher decrease in bacterial counts than the first instrument (P < .01). Intergroup comparison revealed no significant difference between NaOCl and saline after the first instrument (P > .05). NaOCl was significantly better than saline after using the largest instrument in the series (P < .01). Conclusions: Irrespective of the type of irrigant, an increase in the apical preparation size significantly enhanced root canal disinfection. The disinfecting benefit of NaOCl over saline was significant at large apical preparation sizes. HighlightsThe influence of apical enlargement on bacterial reduction was evaluated.Irrigation was done with either sodium hypochlorite (NaOCl) or saline.The larger the apical preparation size, the greater the bacterial reduction.NaOCl was superior to saline only at large apical preparation sizes.

Bacterial diversity in medication-related osteonecrosis of the jaw

OBJECTIVE The aim was to study the association between microflora and medication-related osteonecrosis of the jaw (MRONJ) by using culture-independent molecular techniques to detect bacteria in necrotic bone lesions. STUDY DESIGN Included were 18 consecutive patients with MRONJ, 10 with osteoporosis and 8 cancer patients. Bone biopsies were retrieved from the center of the necrotic bone and from visually healthy bone, and 16 S rRNA gene fragments from bacterial DNA were amplified with polymerase chain reaction. RESULTS The study revealed a diversity of bacteria represented by 16 S rRNA sequences in all the necrotic bone samples and in 60% of the visually healthy bone. Eight dominating taxa groups were identified at the genus level: Porphyromonas, Lactobacillus, Tannerella, Prevotella, Actinomyces, Treponema, Streptococcus, and Fusobacterium. CONCLUSIONS The necrotic bone lesions contained mainly anaerobic bacteria, representative of periodontal microflora, suggesting that a periodontal infection in combination with antiresorptive treatment could initiate osteonecrosis.

Polymorphisms in the interleukin-10 gene and chronic periodontitis in patients with atherosclerotic and aortic aneurysmal vascular diseases

Background Chronic periodontitis (CP), atherosclerotic and aortic aneurysmal vascular diseases (VD) are chronic inflammatory conditions with multifactorial etiologies, including involvement of predisposing genetic factors. In a previous study, polymorphisms in the gene for the anti-inflammatory interleukin-1 receptor antagonist were associated with CP in patients with VD. Objective This study investigates whether polymorphisms in the gene for the anti-inflammatory interleukin-10 (IL10) could be related to CP in the same manner. Methods Seventy-two patients with VD of whom 35 had CP were genotyped for single nucleotide polymorphisms (SNPs) in the IL10 −592 (rs1800872), −819 (rs1800871), and −1,082 (rs1800896) gene by Taqman rtPCR method and by DNA sequencing. Results The C alleles and C/C genotypes of IL10 −592 and IL10 −819 frequencies were significantly higher, while the frequencies of the IL10 −592 (C/A) and IL10 −819 (C/T) heterozygote genotypes were significantly lower in the VD group with CP compared to those without CP. The IL10 haplotype ATA frequency (−1,082, −819, −592) showed a trend to a significant difference between the two groups indicating protection against CP. Conclusions Taken together, our findings suggest an independent association of genetic polymorphisms in the IL-10 gene locus with CP in patients with VD. Development of CP and the implications on vascular disease emphasize the importance of early detection and adequate treatment of periodontitis among these patients.

Polymorphisms in the Interleukin-1 Gene Locus and Chronic Periodontitis in Patients with Atherosclerotic and Aortic Aneurysmal Vascular Diseases

Chronic periodontitis (CP) and atherosclerotic and aortic aneurysmal vascular diseases (VD) are inflammatory conditions that share a number of predisposing factors. They have a complex genetic heritability and may share genetic risk factors, but a well‐defined relationship is still not determined. In addition, distinct genetic patterns of predisposition have been associated with these diseases. Here, we investigated the association of polymorphisms in the IL‐1 gene locus with CP in a case–case study analysing VD patients with or without CP. Seventy‐four patients with VD of whom 36 had CP were genotyped for single nucleotide polymorphisms in the IL1A ‐889 (rs1800587), IL1B +3954 (rs1143634) and IL1B at −511 (rs16944) genes and for VNTR polymorphisms in the IL1RN gene. A significantly higher frequency (17%) for allele 1 (four repeats) of the IL1RN VNTR gene was found among the VD patients with CP compared to those without CP. In addition, the frequency of the IL1RN VNTR genotypes 1/1 (4/4 repeats) and 2/2 (2/2 repeats) were significantly higher and lower, respectively, in VD patients with CP. These findings suggest an association of genetic polymorphisms in the IL1‐gene locus with risk for CP in patients with VD. The carriage of the risk genotypes, the development and the subsequent influence of CP on systemic health may constitute an additional burden in the pathogenesis of VD. This emphasizes the importance of effective periodontal treatment in patients with VD.

Juvenile idiopathic arthritis and rheumatoid arthritis: bacterial diversity in temporomandibular joint synovial fluid in comparison with immunological and clinical findings

Temporomandibular joint (TMJ) involvement in juvenile idiopathic arthritis (JIA) occurs in up to 80% of affected children. The purpose of this study was to investigate the presence of bacterial DNA in synovial fluid, and to compare this with clinical and immunological findings in children with JIA, adults with persistent JIA, and adults with rheumatoid arthritis, in order to detect whether bacteria contribute to inflammation in TMJ arthritis. Synovial fluid and skin swab samples were collected from 30 patients (54 TMJs). Bacterial detection was performed using 16S rRNA pyrosequencing. Bacterial DNA was detected in 31 TMJs (57%) in 19 patients (63%). A positive statistically significant correlation was registered between bacterial DNA detected in TMJ synovial fluid and the following factors: total protein concentration in synovial fluid, interleukin 1β, tumour necrosis factor alpha, adrenocorticotropic hormone, and adiponectin, as well as the duration of the general medical disease. Fourteen different bacterial species were detected in synovial fluid. Bacterial DNA in TMJ synovial fluid without contamination was detected in more than 50% of the patients. Studies are needed to evaluate the consequences of this bacterial DNA in synovial fluid with regard to TMJ arthritis.