Patient understanding of genetic information influences reproductive decision making in retinoblastoma.

BACKGROUND: Retinoblastoma is the most common malignant tumour of the eye in childhood, with nearly all bilateral tumours and around 17% to 18% of unilateral tumours due to an oncogenic mutation in the RB1 gene in the germline. Genetic testing enables accurate risk assessment and optimal clinical management for the affected individual, siblings, and future offspring. MATERIAL AND METHODS: We carried out the first UK-wide audit of understanding of genetic testing in individuals with retinoblastoma. A total of 292 individuals aged 16 to 45 years were included. RESULTS: Patients with bilateral disease were significantly more likely to understand the implications of retinoblastoma for siblings and children. There was a significant association between not knowing the results of genetic testing or not understanding the implications and not having children, particularly in women. Surprisingly, this was also true for individuals treated for unilateral disease with a low risk of retinoblastoma for their offspring. CONCLUSION: We are concerned that individuals may be making life choices based on insufficient information regarding risks of retinoblastoma and reproductive options. We suggest that improvement in transition care is needed to enable individuals to make informed reproductive decisions and to ensure optimal care for children born at risk of retinoblastoma.

Collagen-like peptide sequences inhibit bacterial invasion of root dentine.

AIM: To investigate the effects of peptides derived from the sequence of collagen to inhibit penetration of human or bovine dentine by species of streptococci and enterococci. METHODOLOGY: Blocks of human or bovine root dentine were infected for 14 days with bacterial cultures, in the presence or absence of various collagen-like peptide sequences. Invasion of dentinal tubules was determined from microscopic images of histochemically stained dentine thin sections. Extent of invasion was expressed as tubule invasion index (TI), or tubule invasion factor (TIF) which, in addition to the density of invasion, took into account the depth of invasion. Data were analysed by two-way anova. RESULTS: Streptococcus gordonii, Streptococcus mutans and Enterococcus faecalis were associated with heavy invasion (TI >2.5, TIF >4) of human or bovine root dentinal tubules, with E. faecalis being the most penetrative. Incorporation of peptides Gly-Pro-Ala or Gly-Pro-Hyp into the in vitro model system significantly reduced (P < 0.05) dentine invasion by the three species of highly invasive organisms. Inhibition of bacterial invasion by the peptides was dose dependent, and the peptides did not inhibit bacterial growth in culture. CONCLUSION: Specific collagen-like peptide sequences inhibited the invasion of dentine in vitro by a range of oral bacteria. The peptides likely act as competitive inhibitors blocking bacterial collagen receptors and could potentially allow for target-specific control of dentine infections.

The effects of different orthodontic appliances upon microbial communities.

OBJECTIVES: Orthodontic appliances can promote accumulation of dental plaque, with associated enamel decalcification or gingival inflammation. The aim of this study was to examine longer-term microbiological changes during orthodontic treatment with fixed appliances. MATERIALS AND METHODS: Twenty-four orthodontic patients aged 11-14 years undergoing fixed appliance therapy were recruited into the study. Each was randomized for cross-mouth assignment of molar bands and bonded molar tubes to contralateral quadrants of the mouth. All patients received self-ligating brackets, but again using randomization, one upper lateral incisor bracket (left or right) also received an elastomeric ligature. Plaque samples from the molars and upper lateral incisors were obtained at intervals during treatment and up to 1 year after appliance removal. Denaturing gradient gel electrophoresis and 16S rDNA microarray were used to compare plaque microbial fingerprints. RESULTS: Plaque populations changed within 3 months of commencing treatment at all sites. The greatest differences in plaque composition were seen with self-ligating brackets with an elastomeric ligature. Post-treatment plaque associated with both types of molar attachment contained increased levels of periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Eubacterium nodatum, while Campylobacter rectus, Parvimonas micra, and Actinomyces odontolyticus were also elevated with bonds. CONCLUSIONS: The results suggest that orthodontic treatment may cause sustained changes in plaque microbiotas and that molar bond-associated plaque may have raised disease potential.

Gene expression profiling identifies different sub-types of retinoblastoma.

BACKGROUND: Mutation of the RB1 gene is necessary but not sufficient for the development of retinoblastoma. The nature of events occurring subsequent to RB1 mutation is unclear, as is the retinal cell-of-origin of this tumour. METHODS: Gene expression profiling of 21 retinoblastomas was carried out to identify genetic events that contribute to tumorigenesis and to obtain information about tumour histogenesis. RESULTS: Expression analysis showed a clear separation of retinoblastomas into two groups. Group 1 retinoblastomas express genes associated with a range of different retinal cell types, suggesting derivation from a retinal progenitor cell type. Recurrent chromosomal alterations typical of retinoblastoma, for example, chromosome 1q and 6p gain and 16q loss were also a feature of this group, and clinically they were characterised by an invasive pattern of tumour growth. In contrast, group 2 retinoblastomas were found to retain many characteristics of cone photoreceptor cells and appear to exploit the high metabolic capacity of this cell type in order to promote tumour proliferation. CONCLUSION: Retinoblastoma is a heterogeneous tumour with variable biology and clinical characteristics.

Second and subsequent tumours among 1927 retinoblastoma patients diagnosed in Britain 1951-2004.

BACKGROUND: Retinoblastoma is an eye tumour of childhood that occurs in heritable and non-heritable forms. In the heritable form, there is a predisposition to the development of non-ocular subsequent primary tumours (SPTs). METHODS: This study included 1927 retinoblastoma patients diagnosed in Britain from 1951 to 2004. Ascertainment was through the (UK) National Registry of Childhood Tumours; cases were followed-up for the occurrence of SPTs. Standardised incidence ratios (SIRs) were calculated. RESULTS: We identified 169 SPTs in 152 patients. The SIR analysis included 145 SPTs with cancer registrations from the years 1971 to 2009. These tumours occurred in 132 patients: 112 of the 781 heritable and 20 of the 1075 (presumed) non-heritable cases under surveillance at the start of this period developed at least one registered SPT. The SIRs for all tumours combined were 13.7 (95% confidence interval 11.3-16.5) in heritable cases and 1.5 (0.9-2.3) in non-heritable cases. The main types of SPT in the heritable cases were leiomyosarcoma, (31 cases; SIR 1018.7 (692.2-1446.0)), osteosarcoma (26 cases; SIR 444.6 (290.4-651.4)), and skin melanoma (12 cases; SIR 18.6 (9.6-32.4)). CONCLUSION: The risk of SPTs in heritable retinoblastoma is extremely high. This has important implications for the clinical follow-up and counselling of survivors and their families.

Retinoblastoma: From genes to patient care.

Retinoblastoma is the most common paediatric neoplasm of the retina, and one of the earliest model of cancer genetics since the identification of the master tumour suppressor gene RB1. Tumorigenesis has been shown to be driven by pathogenic variants of the RB1 locus, but also genomic and epigenomic alterations outside the locus. The increasing knowledge on this "mutational landscape" is used in current practice for precise genetic testing and counselling. Novel methods provide access to pre-therapeutic tumour DNA, by isolating cell-free DNA from aqueous humour or plasma. This is expected to facilitate assessment of the constitutional status of RB1, to provide an early risk stratification using molecular prognostic markers, to follow the response to the treatment in longitudinal studies, and to predict the response to targeted therapies. The aim of this review is to show how molecular genetics of retinoblastoma drives diagnosis, treatment, monitoring of the disease and surveillance of the patients and relatives. We first recap the current knowledge on retinoblastoma genetics and its use in every-day practice. We then focus on retinoblastoma subgrouping at the era of molecular biology, and the expected input of cell-free DNA in the field.

Incidence and survival of childhood bone cancer in northern England and the West Midlands, 1981-2002.

There is a paucity of population-based studies examining incidence and survival trends in childhood bone tumours. We used high quality data from four population-based registries in England. Incidence patterns and trends were described using Poisson regression. Survival trends were analysed using Cox regression. There were 374 cases of childhood (ages 0-14 years) bone tumours (206 osteosarcomas, 144 Ewing sarcomas, 16 chondrosarcomas, 8 other bone tumours) registered in the period 1981-2002. Overall incidence (per million person years) rates were 2.63 (95% confidence interval (CI) 2.27-2.99) for osteosarcoma, 1.90 (1.58-2.21) for Ewing sarcoma and 0.21 (0.11-0.31) for chondrosarcoma. Incidence of Ewing sarcoma declined at an average rate of 3.1% (95% CI 0.6-5.6) per annum (P=0.04), which may be due to tumour reclassification, but there was no change in osteosarcoma incidence. Survival showed marked improvement over the 20 years (1981-2000) for Ewing sarcoma (hazard ratio (HR) per annum=0.95 95% CI 0.91-0.99; P=0.02). However, no improvement was seen for osteosarcoma patients (HR per annum=1.02 95% CI 0.98-1.05; P=0.35) over this time period. Reasons for failure to improve survival including potential delays in diagnosis, accrual to trials, adherence to therapy and lack of improvement in treatment strategies all need to be considered.

Langerhans cell histiocytosis associated with breast carcinoma successfully treated with topical imiquimod.

Langerhans cell histiocytosis (LCH) encompasses a group of disorders characterized by the proliferation and infiltration of Langerhans cells within internal organs and/or skin. There is often multiorgan involvement; isolated cutaneous LCH is less common. The aetiology of cutaneous LCH remains uncertain, and debate remains as to whether LCH represents a neoplastic condition or is simply reactive. We report a 53-year-old woman who developed isolated cutaneous LCH 15 months after being diagnosed with infiltrating ductal carcinoma of the left breast. The LCH was treated with topical imiquimod, resulting in clinical and histological resolution. Our case highlights the rare association between cutaneous LCH and breast carcinoma, and the clinical and histological response that can be achieved with topical imiquimod. After a diagnosis of LCH, patients require long-term follow-up, due to the risk of recurrence and/or development of a subsequent malignancy.

Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis.

Porphyromonas gingivalis, a gram-negative anaerobe, is a major etiological agent in the initiation and progression of severe forms of periodontal disease. An opportunistic pathogen, P. gingivalis can also exist in commensal harmony with the host, with disease episodes ensuing from a shift in the ecological balance within the complex periodontal microenvironment. Colonization of the subgingival region is facilitated by the ability to adhere to available substrates such as adsorbed salivary molecules, matrix proteins, epithelial cells, and bacteria that are already established as a biofilm on tooth and epithelial surfaces. Binding to all of these substrates may be mediated by various regions of P. gingivalis fimbrillin, the structural subunit of the major fimbriae. P. gingivalis is an asaccharolytic organism, with a requirement for hemin (as a source of iron) and peptides for growth. At least three hemagglutinins and five proteinases are produced to satisfy these requirements. The hemagglutinin and proteinase genes contain extensive regions of highly conserved sequences, with posttranslational processing of proteinase gene products contributing to the formation of multimeric surface protein-adhesin complexes. Many of the virulence properties of P. gingivalis appear to be consequent to its adaptations to obtain hemin and peptides. Thus, hemagglutinins participate in adherence interactions with host cells, while proteinases contribute to inactivation of the effector molecules of the immune response and to tissue destruction. In addition to direct assault on the periodontal tissues, P. gingivalis can modulate eucaryotic cell signal transduction pathways, directing its uptake by gingival epithelial cells. Within this privileged site, P. gingivalis can replicate and impinge upon components of the innate host defense. Although a variety of surface molecules stimulate production of cytokines and other participants in the immune response, P. gingivalis may also undertake a stealth role whereby pivotal immune mediators are selectively inactivated. In keeping with its strict metabolic requirements, regulation of gene expression in P. gingivalis can be controlled at the transcriptional level. Finally, although periodontal disease is localized to the tissues surrounding the tooth, evidence is accumulating that infection with P. gingivalis may predispose to more serious systemic conditions such as cardiovascular disease and to delivery of preterm infants.

Is postal follow-up of childhood cancer survivors worthwhile?

BACKGROUND: Childhood cancer survival has increased over the last 30 years, but long-term effects necessitate continued monitoring of survivors. Since not all of them attend follow-up clinics, this study assesses the efficacy of obtaining information from general practitioners (GPs) through a 5-year rolling postal program. PROCEDURE: Survivors were included who had been diagnosed with a malignancy in the West Midlands since 1957 and were not attending central long-term follow-up clinics. RESULTS: One thousand twenty-seven patients were followed up between 1993 and 2004. Replies were received on 903 (88% response). There were 44 subsequent malignancies and 42 deaths. No medical problems were reported in 341/935 patients (36.5%); in the other 594 endocrine effects were the most common, with visual effects the biggest single problem. Brain tumor survivors had the largest proportion of problems. CONCLUSIONS: The response rate and information quality achieved show that this method of follow-up is feasible, in cases of discharged or defaulting patients. These data will complement those derived from hospital-based follow-up studies, to give a broader understanding of the spectrum of late effects experienced by survivors and may inform the development of specific long-term follow-up protocols.

Interspecies dynamics among bacteria associated with canine periodontal disease.

The etiology and pathogenic mechanisms associated with canine periodontal disease are less well understood than the disease in humans. In this study we have reconstructed defined consortia biofilms in vitro of microorganisms identified as prevalent in a same-breed cohort of dogs with or without periodontal disease. Frederiksenia canicola and Neisseria canis were selected as potential early colonizers of salivary pellicle, and Fusobacterium nucleatum and Porphyromonas gulae were included as high incidence canine oral bacteria. N. canis formed a biofilm substratum under aerobic conditions, but was unable to tolerate anaerobic conditions. Fr. canicola exhibited synergistic biofilm growth with Po. gulae under anaerobic conditions, but displayed an antagonistic relationship with Fu. nucleatum. However, strong co-adhesion between Fu. nucleatum and Po. gulae was able to overcome the inhibitory effects of Fr. canicola to facilitate three-species biofilm formation. Parvimonas micra, an anaerobic, asaccharolytic Gram-positive coccus found only under disease conditions in vivo, was able to form biofilms in conjunction with Fr. canicola and Po. gulae. Furthermore, the specific proteolytic activities of biofilms containing Fr. canicola and Po. gulae or Fu. nucleatum and Po. gulae were increased several-fold upon the addition of Pa. micra. This suggests that anaerobic cocci such as Pa. micra might provide a catalyst for progressive tissue destruction, inflammation and alveolar bone loss in canine periodontal disease, in keeping with the keystone-pathogen hypothesis.

Silver doped titanium dioxide nanoparticles as antimicrobial additives to dental polymers.

OBJECTIVE: The objectives of this in vitro study were to produce a filled resin containing Ag-TiO2 filler particles and to test its antibacterial properties. METHODS: Ag-TiO2 particles were manufactured using the ball milling method and incorporated into an epoxy resin using a high speed centrifugal mixer. Using UV/vis spectrophotometry investigations were performed to assess how the photocatalytic properties of the Ag-TiO2 particles are affected when encased in resin. Adopting the bacteria colony counting technique, the antibacterial properties of Ag-TiO2 particles and Ag-TiO2 containing resins were assessed using Streptococcus mutans under varying lighting conditions. RESULTS: Ag doping of TiO2 results in a band gap shift towards the visible spectrum enabling Ag-TiO2 to exhibit photocatalytic properties when exposed to visible light. Small quantities of Ag-TiO2 were able to produce a bactericidal effect when in contact with S. mutans under visible light conditions. When incorporated into the bulk of an epoxy resin, the photocatalytic properties of the Ag-TiO2 particles were significantly reduced. However, a potent bactericidal effect was still achieved against S. mutans. SIGNIFICANCE: Ag-TiO2 filled resin shows promising antimicrobial properties, which could potentially be used clinically.

Interactions between Streptococcus oralis, Actinomyces oris, and Candida albicans in the development of multispecies oral microbial biofilms on salivary pellicle.

The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S. oralis and A. oris interact with each other and with C. albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence in situ hybridization (FISH). Actinomyces oris and S. oralis formed robust dual-species biofilms, or three-species biofilms with C. albicans. The bacterial components tended to dominate the lower levels of the biofilms while C. albicans occupied the upper levels. Non-fimbriated A. oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C. albicans. Biofilm growth and hyphal filament production by C. albicans was enhanced by S. oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C. albicans persistence at multiple oral cavity sites.

Transcriptome analysis of Streptococcus gordonii Challis DL1 indicates a role for the biofilm-associated fruRBA operon in response to Candida albicans.

Multiple levels of interkingdom signaling have been implicated in maintaining the ecological balance between Candida albicans and commensal streptococci to assure a state of oral health. To better understand the molecular mechanisms involved in the initial streptococcal response to the presence of C. albicans that can initiate oral surface colonization and biofilm formation, hypha-forming cells were incubated with Streptococcus gordonii cells for 30 min to assess the streptococcal transcriptome response. A genome-wide microarray analysis and quantitative polymerase chain reaction validation of S. gordonii transcripts identified a number of genes, the majority of which were involved in metabolic functions that were differentially expressed in the presence of hyphae. The fruR, fruB, and fruA genes encoding the transcriptional regulator, fructose-1-phosphate kinase, and fructose-specific permease, respectively, of the phosphoenolpyruvate-dependent fructose phosphotransferase system, were consistently upregulated. An S. gordonii mutant in which these genes were deleted by allelic replacement formed an architecturally distinct, less robust biofilm with C. albicans than did parental strain cells. Complementing the mutant with plasmid borne fruR, fruB, and fruA genes caused phenotype reversion, indicating that the genes in this operon played a role in dual-species biofilm formation. This genome-wide analysis of the S. gordonii transcriptional response to C. albicans has identified several genes that have potential roles in interkingdom signaling and responses.

Transcriptional landscape of trans-kingdom communication between Candida albicans and Streptococcus gordonii.

Recent studies have shown that the transcriptional landscape of the pleiomorphic fungus Candida albicans is highly dependent upon growth conditions. Here using a dual RNA-seq approach we identified 299 C. albicans and 72 Streptococcus gordonii genes that were either upregulated or downregulated specifically as a result of co-culturing these human oral cavity microorganisms. Seventy-five C. albicans genes involved in responses to chemical stimuli, regulation, homeostasis, protein modification and cell cycle were significantly (P ≤ 0.05) upregulated, whereas 36 genes mainly involved in transport and translation were downregulated. Upregulation of filamentation-associated TEC1 and FGR42 genes, and of ALS1 adhesin gene, concurred with previous evidence that the C. albicans yeast to hypha transition is promoted by S. gordonii. Increased expression of genes required for arginine biosynthesis in C. albicans was potentially indicative of a novel oxidative stress response. The transcriptional response of S. gordonii to C. albicans was less dramatic, with only eight S. gordonii genes significantly (P ≤ 0.05) upregulated at least two-fold (glpK, rplO, celB, rplN, rplB, rpsE, ciaR and gat). The expression patterns suggest that signals from S. gordonii cause a positive filamentation response in C. albicans, whereas S. gordonii appears to be transcriptionally less influenced by C. albicans.

Adherence and accumulation of oral streptococci.

Oral streptococci adhere to human salivary components and coadhere with specific partner oral bacteria. These interactions may favour the ordered development of plaque communities. The primary sequences of several streptococcal polypeptide adhesins are conserved, indicating that similar colonization mechanisms may have evolved. Critical amino acid changes within binding domains of adhesins might account for species- and site-specific adherence and accumulation.

Streptococcus gordonii DL1 adhesin SspB V-region mediates coaggregation via receptor polysaccharide of Actinomyces oris T14V.

Streptococcus gordonii SspA and SspB proteins, members of the antigen I/II (AgI/II) family of Streptococcus adhesins, mediate adherence to cysteine-rich scavenger glycoprotein gp340 and cells of other oral microbial species. In this article we investigated further the mechanism of coaggregation between S. gordonii DL1 and Actinomyces oris T14V. Previous mutational analysis of S. gordonii suggested that SspB was necessary for coaggregation with A. oris T14V. We have confirmed this by showing that Lactococcus lactis surrogate host cells expressing SspB coaggregated with A. oris T14V and PK606 cells, while L. lactis cells expressing SspA did not. Coaggregation occurred independently of expression of A. oris type 1 (FimP) or type 2 (FimA) fimbriae. Polysaccharide was prepared from cells of A. oris T14V and found to contain 1,4-, 4,6- and 3,4-linked glucose, 1,4-linked mannose, and 2,4-linked galactose residues. When immobilized onto plastic wells this polysaccharide supported binding of L. lactis expressing SspB, but not binding of L. lactis expressing other AgI/II family proteins. Purified recombinant NAVP region of SspB, comprising amino acid (aa) residues 41-847, bound A. oris polysaccharide but the C-domain (932-1470 aa residues) did not. A site-directed deletion of 29 aa residues (Δ691-718) close to the predicted binding cleft within the SspB V-region ablated binding of the NAVP region to polysaccharide. These results infer that the V-region head of SspB recognizes an actinomyces polysaccharide ligand, so further characterizing a lectin-like coaggregation mechanism occurring between two important primary colonizers.

A mutation in the V1 domain of keratin 5 causes epidermolysis bullosa simplex with mottled pigmentation.

Epidermolysis bullosa simplex with mottled pigmentation (EBS-MP; MIM no 131960) is an autosomal dominant disorder characterized by skin blistering at acral sites, punctate palmo-plantar hyperkeratoses, and mottled pigmentation of the trunk and proximal extremities. Histologically and ultrastructurally, the blistering in EBS-MP closely resembles that found in other EBS subtypes. This is consistent with a disorder of the basal keratinocyte cytoskeleton, in which several groups have found disease-causing mutations within the central rod domains of keratins 5 and 14. We have identified a C --> T transition at base position 71 of K5 causing a P24L substitution in a sporadic case of EBS-MP. Recently, this same mutation was identified in two unrelated families with EBS-MP.

Cell surface protein receptors in oral streptococci.

Streptococci have a vast repertoire of adherence properties which include binding to human tissue components, epithelial cells and to other bacterial cells. These interactions are determined by the expression of cell-surface receptors some of which are species-specific. In the oral streptococci, two families of surface protein receptors with highly conserved amino acid sequences have been identified. The antigen I/II family of polypeptides are wall-associated high molecular mass proteins (158-166 kDa) with several binding functions that may be attributed to different domains of the receptor molecules. The LraI family of polypeptides are surface-associated lipoproteins (32-33 kDa) involved in adherence of streptococci to salivary glycoprotein pellicle and to oral Actinomyces. A region of amino acid sequence similarity is evident amongst members of the two protein families in Streptococcus gordonii. Ligand-binding specificities of these receptor polypeptides may account for species-specific adherence and site-directed colonization of streptococci within the human oral cavity.

Yeast-specific DNA probes and their application for the detection of Candida albicans.

Two DNA fragments cloned from the genome of Candida albicans ATCC 10261 may be useful in the rapid diagnosis of disseminated candidosis. One sequence (probe EOB1) was specific for C. albicans (positive hybridisation with 45 strains tested). The second sequence (probe EOB2) detected C. albicans, as well as five other pathogenic Candida spp. and Saccharomyces cerevisiae, but did not react with human or bacterial DNA. Both probes were repetitive sequences in the genome of C. albicans. Probe EOB1 was used to detect, without DNA amplification, 500 C. albicans yeast cells in 1 ml of human blood.