Histological profile of tumours from MYCN transgenic mice.

BACKGROUND: MYCN is the most commonly amplified gene in human neuroblastomas. This proto-oncogene has been overexpressed in a mouse model of the disease in order to explore the role of MYCN in this tumour. AIMS: To report the histopathological features of neuroblastomas from MYCN transgenic mice. METHODS: 27 neuroblastomas from hemizygous transgenic mice and four tumours from homozygous mice were examined histologically; Ki67 and MYCN immunocytochemistry was performed in 24 tumours. RESULTS: Tumours obtained from MYCN transgenic mice resembled human neuroblastomas, displaying many of the features associated with stroma-poor neuroblastoma, including heterogeneity of differentiation (but no overt ganglionic differentiation was seen), low levels of Schwannian stroma and a high mitosis karyorrhexis index. The tumours had a median Ki67 labelling index of 70%; all tumours expressed MYCN with a median labelling index of 68%. The most striking difference between the murine and human neuroblastomas was the presence of tingible body macrophages in the transgenic mouse tumours reflecting high levels of apoptosis. This has not previously been described in human or other murine neuroblastoma models. CONCLUSIONS: These studies highlight the histological similarities between tumours from MYCN transgenic mice and human neuroblastomas, and reaffirm their role as a valuable model to study the biology of aggressive human neuroblastoma.

Minimal disease monitoring by QRT-PCR: guidelines for identification and systematic validation of molecular markers prior to evaluation in prospective clinical trials.

Real-time RT-PCR (QRT-PCR) is a sensitive method for the detection of minimal disease (MD) and may improve monitoring of disease status and stratification of patients for therapy. Where tumour-specific mRNAs have not been identified, the selection of which target(s) is(are) optimal for the detection of MD remains a challenge. This reflects the heterogeneity of tumour cells, the stability of mRNAs and low-level of transcription in cells of the normal haemopoietic compartments. The aim of this study was to establish for the first time guidelines for the systematic prioritization of potential markers of MD detected by QRT-PCR prior to evaluation in multicentre prospective clinical outcome studies. We combined microarray analysis, ESTs gene expression profiles, improved probe-sets sequence annotation, and previously described standard operating procedures for QRT-PCR analysis to identify and prioritize potential markers of MD. Using this methodology, we identified 49 potential markers of MD in neuroblastoma (NB), of which 11 were associated with neuronal function. We found that, in addition to TH, Phox2B and DCX mRNA may be useful targets for the detection of MD in children with NB. This same strategy could be exploited to select MD markers of other solid tumours from the large number of potential targets identified by microarray gene expression profiles.

Identification of candidate genes involved in neuroblastoma progression by combining genomic and expression microarrays with survival data.

Identifying genes, whose expression is consistently altered by chromosomal gains or losses, is an important step in defining genes of biological relevance in a wide variety of tumour types. However, additional criteria are needed to discriminate further among the large number of candidate genes identified. This is particularly true for neuroblastoma, where multiple genomic copy number changes of proven prognostic value exist. We have used Affymetrix microarrays and a combination of fluorescent in situ hybridization and single nucleotide polymorphism (SNP) microarrays to establish expression profiles and delineate copy number alterations in 30 primary neuroblastomas. Correlation of microarray data with patient survival and analysis of expression within rodent neuroblastoma cell lines were then used to define further genes likely to be involved in the disease process. Using this approach, we identify >1000 genes within eight recurrent genomic alterations (loss of 1p, 3p, 4p, 10q and 11q, 2p gain, 17q gain, and the MYCN amplicon) whose expression is consistently altered by copy number change. Of these, 84 correlate with patient survival, with the minimal regions of 17q gain and 4p loss being enriched significantly for such genes. These include genes involved in RNA and DNA metabolism, and apoptosis. Orthologues of all but one of these genes on 17q are overexpressed in rodent neuroblastoma cell lines. A significant excess of SNPs whose copy number correlates with survival is also observed on proximal 4p in stage 4 tumours, and we find that deletion of 4p is associated with improved outcome in an extended cohort of tumours. These results define the major impact of genomic copy number alterations upon transcription within neuroblastoma, and highlight genes on distal 17q and proximal 4p for downstream analyses. They also suggest that integration of discriminators, such as survival and comparative gene expression, with microarray data may be useful in the identification of critical genes within regions of loss or gain in many human cancers.

Quantum opacity, the RHIC Hanbury Brown-Twiss puzzle, and the chiral phase transition.

We present a relativistic quantum-mechanical treatment of opacity and refractive effects that allows reproduction of observables measured in two-pion Hanbury Brown-Twiss (HBT) interferometry and pion spectra at RHIC. The inferred emission duration is substantial. The results are consistent with the emission of pions from a system that has a restored chiral symmetry.

Evolutionary implications of pericentromeric gene expression in humans.

Human pericentromeric sequences are enriched for recent sequence duplications. The continual creation and shuffling of these duplications can create novel intron-exon structures and it has been suggested that these regions have a function as gene nurseries. However, these sequences are also rich in satellite repeats which can repress transcription, and analyses of chromosomes 10 and 21 have suggested that they are transcript poor. Here, we investigate the relationship between pericentromeric duplication and transcription by analyzing the in silico transcriptional profiles within the proximal 1.5 Mb of genomic sequence on all human chromosome arms in relation to duplication status. We identify an approximately 5x excess of transcripts specific to cancer and/or testis in pericentromeric duplications compared to surrounding single copy sequence, with the expression of >50% of all transcripts in duplications being restricted to these tissues. We also identify an approximately 5x excess of transcripts in duplications which contain large quantities of interspersed repeats. These results indicate that the transcriptional profiles of duplicated and single copy sequences within pericentromeric DNA are distinct, suggesting that pericentromeric instability is unlikely to represent a common route for gene creation but may have a disproportionate effect upon genes whose function is restricted to the germ line.

Staphylococcal species in the oral cavity from patients in a regional burns unit.

The aim of this study was to perform a quantitative and qualitative analysis of oral carriage of staphylococci in a range of oral specimens from patients admitted to a regional burns unit. The study recruited 28 patients and reasons for admittance were: burns (46%), skin grafting (39%), lacerations (7%), scalding (4%) and necrotizing fasciitis (4%). No patient had smoke inhalation injuries or trauma to the oro-pharynx. There were five patients from whom methicillin-sensitive S. aureus (MRSA) could be detected in oral specimens. For three patients only the wound and oral specimens were positive for MRSA. In one patient only the oral specimens were positive for MRSA. There were five patients from whom methicillin-sensitive S. aureus (MRSA) could be detected in the oral specimens. In one patient only the oral specimens were positive for MSSA. Staphylococci could be recovered from the dental plaque, denture and toothbrush specimens with a mean count of 1.1 x 10(4)cfu/mL (range 20-5.3 x 10(4)), 5.4 x 10(3) (range 40-2.1 x 10(4)) and 264 cfu/mL (range 20-500), respectively. Both MSSA and MRSA could be recovered from these specimen types. In one patient only the toothbrush was positive for MRSA and all other oral specimens were negative. This study suggests that staphylococci are not infrequent colonizers of the oral cavity, and that this site may serve as a potential reservoir for transmission to other body sites.

High prevalence of non-albicans yeasts and detection of anti-fungal resistance in the oral flora of patients with advanced cancer.

Oral fungal infections frequently develop in individuals with advanced cancer. This study examined the oral mycological flora of 207 patients receiving palliative care for advanced malignant disease. Demographic details and a clinical history were documented from each participant. A tongue swab was collected and cultured on CHROMAgar Candida (CHROMAgar Paris, France). All yeasts were identified by germ tube test, API ID 32C profiles and, for Candida dubliniensis, by species-specific PCR. Susceptibility to fluconazole and itraconazole was determined by a broth microdilution assay according to the National Committee for Clinical Laboratory Standards (NCCLS). At time of sampling, 54 (26%) of the 207 subjects had clinical evidence of a fungal infection and yeasts were isolated from 139 (67%) individuals. In total, 194 yeasts were isolated, of which 95 (49%) were Candida albicans. There was a high prevalence of Candidia glabrata (47 isolates) of which 34 (72%) were resistant to both fluconazole and itraconazole. All nine isolates of C. dubliniensis recovered were susceptible to both azoles. No relationship was established between anti-fungal usage in the preceding three months and the presence of azole resistant yeasts. This study of patients with advanced cancer has demonstrated a high incidence of oral colonization with non-C. albicans yeasts, many of which had reduced susceptibility to fluconazole and itraconazole. The role of improved oral care regimes and novel anti-fungal drugs merits further attention, to reduce the occurrence of fungal infection in these patients.

Staphylococcus aureus in the oral cavity: a three-year retrospective analysis of clinical laboratory data.

OBJECTIVE: A retrospective analysis of laboratory data to investigate the isolation of Staphylococcus aureus from the oral cavity and facial area in specimens submitted to a regional diagnostic oral microbiology laboratory. METHODS: A hand search of laboratory records for a three-year period (1998-2000) was performed for specimens submitted to the regional diagnostic oral microbiology laboratory based at Glasgow Dental Hospital and School. Data were collected from forms where S. aureus was isolated. These data included demographics, referral source, specimen type, methicillin susceptibility and clinical details. RESULTS: For the period 1998-2000, there were 5,005 specimens submitted to the laboratory. S. aureus was isolated from 1,017 specimens, of which 967 (95%) were sensitive to methicillin (MSSA) and 50 (5%) were resistant to methicillin (MRSA). The 1,017 specimens were provided from 615 patients. MRSA was isolated from 37 (6%) of patients. There was an increasing incidence of S. aureus with age, particularly in the >70 years age group. The most common specimen from which MSSA was isolated was an oral rinse (38%) whilst for MRSA isolates this was a tongue swab (28%). The clinical condition most commonly reported for MSSA isolates was angular cheilitis (22%). Erythema, swelling, pain or burning of the oral mucosa was the clinical condition most commonly reported for MRSA isolates (16%). Patients from whom the MSSA isolates were recovered were most commonly (55%) seen in the oral medicine clinic at the dental hospital, whilst patients with MRSA were more commonly seen in primary care settings such as nursing homes, hospices and general dental practice (51%). CONCLUSION: In line with more recent surveys, this retrospective study suggests that S. aureus may be a more frequent isolate from the oral cavity than hitherto suspected. A small proportion of the S. aureus isolates were MRSA. There were insufficient data available to determine whether the S. aureus isolates were colonising or infecting the oral cavity. However, the role of S. aureus in several diseases of the oral mucosa merits further investigation.

Comparison of two selective media for the detection and enumeration of Lactobacilli in human faeces.

The enumeration of faecal bacteria is an important requirement for many studies of bowel health. One approach is the use of selective culture media for the culture and identification of genera or species from faeces. This study compares the culture of Lactobacilli from dilution series of faecal samples from six healthy human volunteers on two commonly used media, LAMVAB and Rogosa agar. Colonies were counted after a 72-h anaerobic incubation at 37 degrees C, and colony morphology recorded by a single observer. DNA was isolated from a representative number of colonies and genus-specific PCR, single-stranded conformation polymorphism (SSCP) and DNA sequencing performed. Total colony counts ranged from <3.00 to 7.48 log(10) cfu/g of faeces for LAMVAB and 5.09 to 7.66 log(10) cfu/g for Rogosa. For each subject, the total colony count was higher on Rogosa than that obtained with LAMVAB agar. SSCP analysis and DNA sequencing indicated that colony morphology was not an accurate predictor of genus identity. Growth of two species, Lactobacillus acidophilus and Lactobacillus gasseri, was not supported on LAMVAB medium. Rogosa agar was more likely to support growth of non-Lactobacillus species. Therefore, neither medium gave a fully accurate representation of the Lactobacilli species present in human faecal samples.

The ecology of Staphylococcus species in the oral cavity.

Whilst the diversity of organisms present in the oral cavity is well accepted, there remains considerable controversy as to whether Staphylococcus spp. play a role in the ecology of the normal oral flora. Surprisingly little detailed work has been performed on the quantitative and qualitative aspects of colonisation or infection either by coagulase-negative staphylococci (CNS) or S. aureus. The latter is especially interesting in the light of present difficulties in eradicating carriage of methicillin-resistant S. aureus (MRSA) from the oropharynx in affected individuals. This paper reviews the current knowledge of staphylococcal colonisation and infection of the oral cavity in health and disease. S. aureus has been isolated from a wide range of infective oral conditions, such as angular cheilitis and parotitis. More recently, a clinical condition classified as staphylococcal mucositis has emerged as a clinical problem in many debilitated elderly patients and those with oral Crohn's disease. Higher carriage rates of both CNS or S. aureus, or both, in patients prone to joint infections raises the interesting possibility of the oral cavity serving as a potential source for bacteraemic spread to compromised joint spaces. In conclusion, there is a surprising paucity of knowledge regarding the role of oral staphylococci in both health and disease. Further work in this area may lead to benefits, such as improved decolonisation regimens for eradication of MRSA and acknowledgement of the mouth as a source of bacteraemic staphylococci.

Molecular cytogenetic definition of 17q translocation breakpoints in neuroblastoma.

BACKGROUND: Unbalanced translocations resulting in the gain of material from 17q are the most common chromosomal changes in neuroblastoma and are associated with poor patient survival, and are established indicators of bad prognosis. PROCEDURE: We have used 13 fluorescent in situ hybridisation probes to map 17q translocation breakpoints in ten neuroblastoma cell lines and 21 primary tumours. RESULTS: At least seven different breakpoints have been identified, all localised within the proximal half of 17q (53-68 cM, 17cen-17q22). CONCLUSION: These results suggest that the dosage of a gene, or genes, in 17q22-qter is responsible for the clinical effects of 17q gain, rather than the disruption of a specific gene.

Comprehensive genetic and histopathologic study reveals three types of neuroblastoma tumors.

PURPOSE: To determine the relationship between multiple genetic features, tumor morphology, and prognosis in neuroblastoma. PATIENTS AND METHODS: The genetic alterations and morphologic features that underpin three histopathologic risk classifications were analyzed in 108 neuroblastoma patients. Tumors were subdivided into four groups based on the three most frequent and prognostically significant genetic alterations (17q gain, 1p deletion, and MYCN amplification), and all other genetic, morphologic, and clinical data were analyzed with respect to these groups. RESULTS: Our analyses identify three nonoverlapping tumor types with distinct genetic and morphologic features, defined here as types 1, 2, and 3. Type 1 tumors show none of the three significant genetic alterations and have good prognosis. Both type 2 (17q gain only or 17q gain and 1p del) and type 3 (17q gain, 1p del, and MYCN amplification) tumors progress. However, these tumor types are distinguished clinically by having significantly different median age at diagnosis and median progression-free survival (PFS). Multivariate analysis indicates that 17q gain is the only independent prognostic factor among all genetic, histopathologic, and clinical factors analyzed. Among histopathologic risk systems, the International Neuroblastoma Pathology Classification was the best predictor of PFS. CONCLUSION: Our results indicate that specific combinations of genetic changes in neuroblastoma tumors contribute to distinct morphologic and clinical features. Furthermore, the identification of two genetically and morphologically distinct types of progressing tumors suggests that possibilities for different therapeutic regimens should be investigated.

Genomic sequence and transcriptional profile of the boundary between pericentromeric satellites and genes on human chromosome arm 10q.

The organization of centromeric heterochromatin has been established in a number of eucaryotes but remains poorly defined in human. Here we present 1025 kb of contiguous human genomic sequence which links pericentromeric satellites to the RET proto-oncogene in 10q11.2 and is presumed to span the transition from centric heterochromatin to euchromatin on this chromosome arm. Two distinct domains can be defined within the sequence. The proximal approximately 240 kb consists of arrays of satellites and other tandem repeats separated by tracts of complex sequence which have evolved by pericentromeric-directed duplication. Analysis of 32 human paralogues of these sequences indicates that most terminate at or within repeat arrays, implicating these repeats in the interchromosomal duplication process. Corroborative PCR-based analyses establish a genome-wide correlation between the distribution of these paralogues and the distribution of satellite families present in 10q11. In contrast, the distal approximately 780 kb contains few tandem repeats and is largely chromosome specific. However, a minimum of three independent intrachromosomal duplication events have resulted in >370 kb of this sequence sharing >90% identity with sequences on 10p. Using computer-based analyses and RT-PCR we confirm the presence of three genes within the sequence, ZNF11/33B, KIAA0187 and RET, in addition to five transcripts of unknown structure. All of these transcribed sequences map distal to the satellite arrays. The boundary between satellite-rich interchromosomally duplicated DNA and chromosome-specific DNA therefore appears to define a transition from pericentromeric heterochromatin to euchromatin on the long arm of this chromosome.

Origins of Staphylococcus epidermidis and Streptococcus oralis causing bacteraemia in a bone marrow transplant patient.

Coagulase-negative staphylococcal bacteraemia in immunocompromised patients is often associated with the use of central venous catheters, while the proposed origin of viridans streptococci causing bacteraemia in this patient group is the oral cavity. This report describes an episode of polymicrobial bacteraemia caused by Staphylococcus epidermidis and Streptococcus oralis followed by several further episodes of S. epidermidis bacteraemia in a 15-year-old boy after bone marrow transplantation. Pulsed-field gel electrophoresis (PFGE) of SmaI chromosomal DNA digests was used to compare blood culture and oral isolates of S. epidermidis and Str. oralis. The results indicated that the mouth was the source of both S. epidermidis and Str. oralis causing the first episode of bacteraemia. PFGE further demonstrated that the central venous catheter was the origin of a second strain of S. epidermidis responsible for subsequent episodes of staphylococcal bacteraemia. Both the oral mucosa and central venous lines should be considered as potential sources of organisms, including coagulase-negative staphylococci, associated with bacteraemia in immunocompromised patients.

Oral carriage of staphylococci in patients with rheumatoid arthritis.

OBJECTIVE: To determine the prevalence of oral staphylococcal carriage in patients with rheumatoid arthritis compared with healthy controls. METHODS: Fifty healthy adults, 25 healthy elderly volunteers and 25 patients with rheumatoid arthritis were studied. An oral rinse, tongue swab and nasal swab were collected for culture on blood agar and a range of selective agars. Isolates of staphylococci were identified and antibiotic sensitivity profiles determined by standard methods. RESULTS: Staphylococci were isolated from the mouths of 94% of the healthy adults, 24% of whom carried Staphylococcus aureus. All the healthy elderly carried oral staphylococci and 36% were colonized with S. aureus. Staphylococci were isolated from 96% of the rheumatoid arthritis patients and this group had the highest carriage rate of S. aureus (56%), significantly higher than the healthy adults (P < 0.05). In all three groups, Staphylococcus epidermidis was isolated from the mouths of > 80%. No methicillin-resistant strains of S. aureus were isolated. CONCLUSION: Oral carriage of S. aureus appears to be common in patients with rheumatoid arthritis and studies of the mouth as a source of infection in septic arthritis would be merited.

Barriers to the use of a diagnostic oral microbiology laboratory by general dental practitioners.

OBJECTIVE: To identify barriers on the use of diagnostic microbiology facilities in general dental practice. DESIGN: A cross-sectional survey using a postal questionnaire. SETTING: Primary/secondary care interface between the diagnostic oral microbiology laboratory, University of Glasgow Dental Hospital and School, Glasgow and dental practitioners within the surrounding health boards, 1998. SUBJECTS: All GDPs (797) within Argyll and Clyde, Ayrshire and Arran, Lanarkshire and Greater Glasgow Health Boards. MAIN OUTCOME MEASURES: The responses were expressed as both absolute and relative frequencies. RESULTS: Responses were received from 430 (55%). The most frequent reason for failure to use the service was lack of information, with more than half of the respondents claiming to be unaware of the facility. Lack of request forms and sampling equipment were also viewed as barriers to using the service. CONCLUSIONS: The laboratory is failing to successfully communicate its role in addressing the growing burden of antibiotic resistance in the community and must be more proactive in encouraging appropriate use and increasing accessibility of the service to GDPs.