Streptococcus constellatus causing bony destruction secondary to odontogenic infection: three rare cases.

Streptococcus constellatus is part of the Str milleri subgroup. It is a commensal organism that is often present in the oral flora, and has been implicated in pyogenic infections of the central nervous system, abdomen, and deep neck spaces. We present three patients within our unit who developed bony destruction in the facial bones and base of the skull after odontogenic infections. Str constellatus, a known oral and gut commensal that may cause atypical presentations in odontogenic abscesses, was cultured in all cases.

RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis.

Oral and oropharyngeal squamous cell carcinoma (OOSCC) have a low survival rate, mainly due to metastasis to the regional lymph nodes. For optimal treatment of these metastases, a neck dissection is required; however, inaccurate detection methods results in under- and over-treatment. New DNA prognostic methylation biomarkers might improve lymph node metastases detection. To identify epigenetically regulated genes associated with lymph node metastases, genome-wide methylation analysis was performed on 6 OOSCC with (pN+) and 6 OOSCC without (pN0) lymph node metastases and combined with a gene expression signature predictive for pN+ status in OOSCC. Selected genes were validated using an independent OOSCC cohort by immunohistochemistry and pyrosequencing, and on data retrieved from The Cancer Genome Atlas. A two-step statistical selection of differentially methylated sequences revealed 14 genes with increased methylation status and mRNA downregulation in pN+ OOSCC. RAB25, a known tumor suppressor gene, was the highest-ranking gene in the discovery set. In the validation sets, both RAB25 mRNA (P = 0.015) and protein levels (P = 0.012) were lower in pN+ OOSCC. RAB25 mRNA levels were negatively correlated with RAB25 methylation levels (P < 0.001) but RAB25 protein expression was not. Our data revealed that promoter methylation is a mechanism resulting in downregulation of RAB25 expression in pN+ OOSCC and decreased expression is associated with lymph node metastasis. Detection of RAB25 methylation might contribute to lymph node metastasis diagnosis and serve as a potential new therapeutic target in OOSCC.

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma.

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.

Detection of HPV-associated oropharyngeal tumours in a 16-year cohort: more than meets the eye.

BACKGROUND: Accurate assessment of the prevalence of the human papilloma virus (HPV) in oropharyngeal tumours (OpSCC) is important because HPV-positive OpSCC are consistently associated with an improved overall survival. Recently, an algorithm has become available that reliably detects clinically relevant HPV in tumour tissue, however, no complete cohorts have been tested. The aim was to determine the prevalence of active high-risk HPV infection in a complete cohort of OpSCC collected over a 16-year period. METHODS: Using a triple algorithm of p16 immunohistochemistry, HPV-BRISH and HPV-PCR, we assessed the prevalence of active HPV infection in all OpSCC diagnosed in our hospital from 1997 to 2012 (n=193) and a random selection of 200 oral tumours (OSCC). RESULTS: Forty-seven OpSCC (24%) were HPVGP PCR-positive; 42 cases were HPV16+, 1 HPV18+, 3 HPV33+ and 1 HPV35+. Brightfield in situ hybridisation did not identify additional HPV-positive cases. Human papilloma virus-associated tumour proportion increased from 13% (1997-2004) to 30% (2005-2012). Human papilloma virus-positivity was an independent predictor for longer disease-specific survival (HR=0.22; 95%CI:0.10-0.47). Only one OSCC was HPV+. CONCLUSIONS: In our cohort, the incidence of HPV-associated OpSCC is low but increasing rapidly. The strict detection algorithm, analysis of disease-specific survival and the complete cohort, including palliatively treated patients, may influence the reported prevalence and prognostic value of HPV in OpSCC.

Lack of claudin-7 is a strong predictor of regional recurrence in oral and oropharyngeal squamous cell carcinoma.

OBJECTIVES: Adequate treatment of oral and oropharyngeal squamous cell carcinoma (OSCC) is dependent on correctly predicting the presence of lymph node metastases. Current methods to diagnose nodal metastases partly result in overtreatment with associated morbidity and undertreatment with decreased disease-free survival. E-cadherin has been studied extensively as potential marker for lymph node metastases. EpCAM and claudin-7 have a functional relationship with E-cadherin, forming a complex that promotes tumourigenicity in vitro. We hypothesize that the co-expression patterns of these related molecules is a better prognostic marker for nodal status and regional recurrences. MATERIALS AND METHODS: We constructed separate tissue microarrays of tumour centre and tumour invasive front of 227 OSCC with complete clinicopathological and follow-up data, including HPV status, and performed immunohistochemistry for these molecules. RESULTS: Lack of E-cadherin and presence of cytoplasmic EpCAM expression in the tumour front were predictive for nodal metastasis, but no co-expression pattern was found clinically relevant. Lack of claudin-7 in the tumour centre was highly and independently predictive for shorter regional disease-free survival (HR=0.19; 95%CI: 0.06-0.62) and disease-specific survival (HR=0.43; 95%CI: 0.21-0.87). High-risk HPV was not associated with any marker. CONCLUSIONS: The expression of E-cadherin and EpCAM, depending on the specific tumour sublocalization, is predictive for nodal status. However, co-expression did not improve the prediction of nodal status, indicating that the proposed in vitro complex is not functional in clinical samples. Additionally, lack of claudin-7 expression in the tumour centre may be used to identify patients with increased risk for regional recurrence.

FADD expression is associated with regional and distant metastasis in squamous cell carcinoma of the head and neck.

AIMS: The Fas-associated death domain gene (FADD) is often overexpressed in squamous cell carcinoma of the head and neck (HNSCC), and is considered to be a driver gene in amplification of the chromosomal 11q13.3 region. Amplification of 11q13.3 is associated with increased metastasis in HNSCC and breast cancer. The aim of this study was to investigate the association between FADD protein expression in advanced-stage HNSCC and clinicopathological features and outcome. METHODS AND RESULTS: Tumour tissues of 177 HNSCC patients uniformly treated with primary surgery and postoperative radiotherapy were collected. FADD expression was assessed on pretreatment tumour biopsies using immunohistochemistry. High FADD expression was detected in 44% of the HNSCC patients. High expression was associated with an increased rate of lymph node metastasis (P = 0.001) and with a shorter distant metastasis-free interval (DMFI) (HR 2.6, 95% CI 1.0-6.7, P = 0.046) when lymph node metastases were present. CONCLUSIONS: Our data show that an increase in FADD expression is associated with a higher incidence of lymph node metastasis at presentation, and with shorter DMFI when lymph node metastases are present. High FADD expression in the primary tumour could be a useful marker to select patients for systemic treatment strategies that reduce the risk of distant metastases.

Tumour infiltration depth ≥4 mm is an indication for an elective neck dissection in pT1cN0 oral squamous cell carcinoma.

Patients with pT1cN0 oral squamous cell carcinomas (OSCC) are generally not treated with a neck dissection (ND). However, in 25% of cN0 patients, nodal metastases become apparent during follow-up. Infiltration depth of the primary tumour has been consistently associated with the presence of nodal metastasis, but proposed cut-off depths for performing a ND vary considerably. The aim of this study was to explore the infiltration depth as predictor for the nodal status and to recommend a cut-off depth for performing a ND. From our database of 351 primary oral carcinomas, we selected all pT1-2 tumours (n=246). Infiltration depth was measured in 212 cases. Neck status was determined by histopathological examination of the dissection specimen, or by at least two years of follow-up. Mean infiltration depth was 5.49 mm (95% CI: 4.86-6.12) in the N0 and 8.40 mm (95% CI: 7.38-9.43) in the N+ group (p<0.001). cN status, lymphovascular invasion and infiltration depth were the only independent predictors for nodal status in multiple logistic regression. ROC-analysis on pT1cN0 tumours resulted in an optimal cut-off for the prediction of the nodal status at a depth of 4.59 mm. This cut-off identified a subgroup of patients at increased risk for nodal metastasis (OR=8.3) and with significantly shorter survival. Tumour infiltration depth is an independent predictor for nodal status in pT1-2 OSCC. In pT1cN0 tumours, a cut-off at 4.59 mm results in the best predictive value. We recommend an infiltration depth of ≥4 mm as an indication to perform a neck dissection in pT1cN0 OSCC.

Exercise adherence in patients with trismus due to head and neck oncology: a qualitative study into the use of the Therabite.

Trismus is a common problem after treatment of head and neck cancer. The Therabite is an effective treatment for trismus. To explore the factors that may influence Therabite exercise adherence, how these interrelate and to provide aims for interventions to increase adherence, the authors conducted a multi-centre, formal-evaluative qualitative retrospective study. 21 patients treated for head-neck cancer were interviewed in semi-structured, in-depth interviews. Internal motivation to exercise, the perceived effect, self-discipline and having a clear exercise goal influenced Therabite exercise adherence positively. Perceiving no effect, limitation in Therabite opening range and reaching the exercise goal or a plateau in mouth opening were negative influences. Pain, anxiety and the physiotherapist could influence adherence both positively and negatively. Based on the results, a model for Therabite exercise adherence was proposed. It is important to signal and assess the factors negatively influencing Therabite adherence, specifically before there is a perceived effect. Research is needed to examine why some patients do not achieve results despite high exercise adherence, to identify effective exercise regimens and to assess proposed interventions aimed to increase Therabite exercise adherence.

Reef-coral proteins as visual, non-destructive reporters for plant transformation.

Recently, five novel fluorescent proteins have been isolated from non-bioluminescent species of reef-coral organisms and have been made available through ClonTech. They are AmCyan, AsRed, DsRed, ZsGreen and ZsYellow. These proteins are valuable as reporters for transformation because they do not require a substrate or external co-factor to emit fluorescence and can be tested in vivo without destruction of the tissue under study. We have evaluated them in a large range of plants, both monocots and dicots, and our results indicate that they are valuable reporting tools for transformation in a wide variety of crops. We report here their successful expression in wheat, maize, barley, rice, banana, onion, soybean, cotton, tobacco, potato and tomato. Transient expression could be observed as early as 24 h after DNA delivery in some cases, allowing for very clear visualization of individually transformed cells. Stable transgenic events were generated, using mannose, kanamycin or hygromycin selection. Transgenic plants were phenotypically normal, showing a wide range of fluorescence levels, and were fertile. Expression of AmCyan, ZsGreen and AsRed was visible in maize T1 seeds, allowing visual segregation to more than 99% accuracy. The excitation and emission wavelengths of some of these proteins are significantly different; the difference is enough for the simultaneous visualization of cells transformed with more than one of the fluorescent proteins. These proteins will become useful tools for transformation optimization and other studies. The wide variety of plants successfully tested demonstrates that these proteins will potentially find broad use in plant biology.

Sequence analysis of the vir-region from Agrobacterium tumefaciens octopine Ti plasmid pTi15955.

The nucleotide sequence of 42 775 bp of the vir-region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955 is reported here. Although the nucleotide sequences of several parts of this region from this or closely related plasmids have been published previously, the present work establishes for the first time the complete arrangement of all the essential virulence genes and their intergenic regions of an octopine Ti plasmid. The disruption of some of the intergenic areas by insertion (IS) elements is typical for the octopine Ti plasmids. Several new ORFs were identified, including ORFs immediately downstream of virD4 and virE2, which probably represent new genes involved in virulence.

Novel genes for disease-resistance breeding.

Plant disease control is entering an exciting period during which transgenic plants showing improved resistance to pathogenic viruses, bacteria, fungi and insects are being developed. This review summarizes the first successful attempts to engineer fungal resistance in crops, and highlights two promising approaches. Biotechnology provides the promise of new integrated disease management strategies that combine modern fungicides and transgenic crops to provide effective disease control for modern agriculture.

Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris.

The pgip-1 gene of Phaseolus vulgaris, encoding a polygalacturonase-inhibiting protein (PGIP), PGIP-1 (P. Toubart, A. Desiderio, G. Salvi, F. Cervone, L. Daroda, G. De Lorenzo, C. Bergmann, A. G. Darvill, and P. Albersheim, Plant J. 2:367-373, 1992), was expressed under control of the cauliflower mosaic virus 35S promoter in tomato plants via Agrobacterium tumefaciens-mediated transformation. Transgenic tomato plants with different expression levels of PGIP-1 were used in infection experiments with the pathogenic fungi Fusarium oxysporum f. sp. lycopersici, Botrytis cinerea, and Alternaria solani. No evident enhanced resistance, compared with the resistance of untransformed plants, was observed. The pgip-1 gene was also transiently expressed in Nicotiana benthamiana with potato virus X (PVX) as a vector. PGIP-1 purified from transgenic tomatoes and PGIP-1 in crude protein extracts of PVX-infected N. benthamiana plants were tested with several fungal polygalacturonases (PGs). PGIP-1 from both plant sources exhibited a specificity different from that of PGIP purified from P. vulgaris (bulk bean PGIP). Notably, PGIP-1 was unable to interact with a homogeneous PG from Fusarium moniliforme, as determined by surface plasmon resonance analysis, while the bulk bean PGIP interacted with and inhibited this enzyme. Moreover, PGIP-1 expressed in tomato and N. benthamiana had only a limited capacity to inhibit crude PG preparations from F. oxysporum f. sp. lycopersici, B. cinerea, and A. solani. Differential affinity chromatography was used to separate PGIP proteins present in P. vulgaris extracts. A PGIP-A with specificity similar to that of PGIP-1 was separated from a PGIP-B able to interact with both Aspergillus niger and F. moniliforme PGs. Our data show that PGIPs with different specificities are expressed in P. vulgaris and that the high-level expression of one member (pgip-1) of the PGIP gene family in transgenic plants is not sufficient to confer general, enhanced resistance to fungi.

Production of the AVR9 elicitor from the fungal pathogen Cladosporium fulvum in transgenic tobacco and tomato plants.

Three constructs were used to study the expression of the avirulence gene Avr9 from the fungal tomato pathogen Cladosporium fulvum in plants. They include pAVIR1, pAVIR2 and pAVIR21, encoding the wild-type AVR9 protein and two hybrid AVR9 proteins containing the signal sequences of the pathogenesis-related proteins PR-S and PR-1a, respectively. Transgenic tobacco plants obtained with the three constructs showed a normal phenotype and produced AVR9 elicitor with the same specific necrosis-inducing activity as the wild-type AVR9 elicitor produced in planta by isolates of C. fulvum containing the Avr9 gene. Level of expression was not correlated with number of T-DNA integrations, but plants homozygous for the Avr9 gene produced more elicitor protein than heterozygous plants. The amino acid sequence of the processed AVR9 peptide present in apoplastic fluid (AF) of pAVIR1 transformed plants producing the wild-type AVR9 elicitor was identical to that of the wild-type AVR9 peptide isolated from C. fulvum-infected tomato leaves. Transgenic Cf0 genotypes of tomato, obtained by transformation with construct pAVIR21, showed a normal phenotype. However, transgenic F1 plants expressing the Avr9 gene, obtained from crossing transgenic Cf0 genotypes with wild-type Cf9 genotypes, showed delayed growth, necrosis and complete plant death indicating that the AVR9 peptide produced in plants carrying the Cf9 gene is deleterious. The necrotic defence response observed in Cf9 genotypes expressing the Avr9 gene support the potential to apply avirulence genes in molecular resistance breeding.

Levels of circulating intercellular adhesion molecule-1 and -3 but not circulating endothelial leucocyte adhesion molecule are increased in patients with rheumatoid vasculitis.

The aim of this study was to investigate whether levels of circulating adhesion molecules reflect vascular inflammation in rheumatoid vasculitis (RV). Levels of circulating intercellular adhesion molecule-1 (cICAM-1), c-ICAM-3 and circulating endothelial leucocyte adhesion molecule (cE-selectin) were determined in 14 patients with RV and compared to 47 patients with rheumatoid arthritis (RA) and 100 healthy donors (HD). Enzyme-linked immunosorbent assays were used to quantify cICAM-1, cICAM-3 and cE-selectin. We found that in RV significantly (P < 0.0001) elevated levels of cICAM-1 and cICAM3, but not cE-selectin, were found when compared with RA patients. Levels > 2 S.D. above the mean level of HD were present for cICAM-1, cICAM-3 and cE-selectin in 57, 71 and 21%, respectively of patients with RV and 2, 21 and 44%, respectively of the RA patients. Increased levels of both cICAM-1 and cICAM-3 were found in 43% of the RV patients and in none of the RA patients. Comparison of the serum levels of patients studied in an active and inactive phase of RV revealed significantly lower levels of cICAM-3 levels in the inactive phase. In conclusion we find that determination of cICAM-1 and cICAM-3 may be useful as a marker of vascular inflammation in patients with RV.

A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity.

A novel chitinase gene of tobacco was isolated and characterized by DNA sequence analysis of a genomic clone and a cDNA clone. Comparative sequence analysis of both clones showed an identity of 94%. The proteins encoded by these sequences do not correspond to any of the previously characterized plant chitinases of classes I-IV and are designated as class V chitinases. Comparison of the chitinase class V peptide sequence with sequences in the Swiss Protein databank revealed significant sequence similarity with bacterial exo-chitinases from Bacillus circulans, Serratia marcescens and Streptomyces plicatus. It was demonstrated that class V chitinase gene expression is induced after treatment of tobacco with different forms of stress, like TMV-infection, ethylene treatment, wounding or ultraviolet irradiation. Two related chitinase class V proteins of 41 and 43 kDa were purified from Samsun NN tobacco leaves inoculated with tobacco mosaic virus. The proteins were purified by Chelating Superose chromatography and gel filtration. In vitro assays demonstrated that class V chitinases have endo-chitinase activity and exhibit antifungal activity toward Trichoderma viride and Alternaria radicina. In addition, it was shown that class V chitinase acts synergistically with tobacco class I beta-1,3-glucanase against Fusarium solani germlings.

A novel pathogen- and wound-inducible tobacco (Nicotiana tabacum) protein with antifungal activity.

A novel pathogen- and wound-inducible antifungal protein of 20 kD was purified from tobacco (Nicotiana tabacum) Samsun NN leaves inoculated with tobacco mosaic virus (TMV). The protein, designated CBP20, was purified by chitin-affinity chromatography and gel filtration. In vitro assays demonstrated that CBP20 exhibits antifungal activity toward Trichoderma viride and Fusarium solani by causing lysis of the germ tubes and/or growth inhibition. In addition it was shown that CBP20 acts synergistically with a tobacco class I chitinase against F. solani and with a tobacco class I beta-1,3-glucanase against F. solani and Alternaria radicina. Analysis of the protein and corresponding cDNAs revealed that CBP20 contains an N-terminal chitin-binding domain that is present also in the class I chitinases of tobacco, the putative wound-induced (WIN) proteins of potato, WIN1 and WIN2, and several plant lectins. The C-terminal domain of CBP20 showed high identity with tobacco pathogenesis-related (PR) proteins, PR-4a and PR-4b, tomato PR-P2, and potato WIN1 and WIN2. CBP20 is synthesized as a preproprotein, which is processed into the mature protein by the removal of an N-terminal signal peptide and a C-terminal propeptide, most likely involved in the vacuolar targeting of the protein. The intracellular localization of CBP20 and its induction upon TMV infection and wounding indicate that CBP20 is the first class I PR-4 type protein purified.

Only Specific Tobacco (Nicotiana tabacum) Chitinases and [beta]-1,3-Glucanases Exhibit Antifungal Activity.

Different isoforms of chitinases and [beta]-1,3-glucanases of tobacco (Nicotiana tabacum cv Samsun NN) were tested for their antifungal activities. The class I, vacuolar chitinase and [beta]-1,3-glucanase isoforms were the most active against Fusarium solani germlings, resulting in lysis of the hyphal tips and in growth inhibition. In additon, we observed that the class I chitinase and [beta]-1,3-glucanase acted synergistically. The class II isoforms of the two hydrolases exhibited no antifungal activity. However, the class II chitinases showed limited growth inhibitory activity in combination with higher amounts of class I [beta]-1,3-glucanase. The class II [beta]-1,3-glucanases showed no inhibitory activity in any combination. In transgenic tobacco plants producing modified forms of either a class I chitinase or a class I [beta]-1,3-glucanase, or both, these proteins were targeted extracellularly. Both modified proteins lack their C-terminal propeptide, which functions as a vacuolar targeting signal. Extracellular targeting had no effect on the specific activities of the chitinase and [beta]-1,3-glucanase enzymes. Furthermore, the extracellular washing fluid (EF) from leaves of transgenic plants expressing either of the secreted class I enzymes exhibited antifungal activity on F. solani germlings in vitro comparable to that of the purified vacuolar class I proteins. Mixing EF fractions from these plants revealed synergism in inhibitory activity against F. solani; the mixed fractions exhibited inhibitory activity similar to that of EF from plants expressing both secreted enzymes.

Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and beta-1,3-glucanase in transgenic plants.

The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and beta-1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.

Factors influencing transformation frequency of tomato (Lycopersicon esculentum).

We developed an efficient procedure for transformation and regeneration of L. esculentum cv. Moneymaker from cotyledon explants. The effect of two parameters on the transformation frequency was investigated in detail. The use of feeder layers during cocultivation proved to be critical. In addition, it was found that Agrobacterium strains harbouring a L,L-succinamopine type helper plasmid yielded significantly higher transformation frequencies than those with octopine or nopaline type helper plasmids. The optimized protocol was used to obtain transformation frequencies averaging 9%. Of the plants produced approximately 80% proved to be diploid, of which 67% contained the transgene(s) on a single locus.