Long-range epigenetic silencing of chromosome 5q31 protocadherins is involved in early and late stages of colorectal tumorigenesis through modulation of oncogenic pathways.

Loss of tumour suppressor gene function can occur as a result of epigenetic silencing of large chromosomal regions, referred to as long-range epigenetic silencing (LRES), and genome-wide analyses have revealed that LRES is present in many cancer types. Here we utilize Illumina Beadchip methylation array analysis to identify LRES across 800 kb of chromosome 5q31 in colorectal adenomas and carcinomas (n=34) relative to normal colonic epithelial DNA (n=6). This region encompasses 53 individual protocadherin (PCDH) genes divided among three gene clusters. Hypermethylation within these gene clusters is asynchronous; while most PCDH hypermethylation occurs early, and is apparent in adenomas, PCDHGC3 promoter methylation occurs later in the adenoma-carcinoma transition. PCDHGC3 was hypermethylated in 17/28 carcinomas (60.7%) according to methylation array analysis. Quantitative real-time reverse transcription-polymerase chain reaction showed that PCDHGC3 is the highest expressed PCDH in normal colonic epithelium, and that there was a strong reciprocal relationship between PCDHGC3 methylation and expression in carcinomas (R=-0.84). PCDH LRES patterns are reflected in colorectal tumour cell lines; adenoma cell lines are not methylated at PCDHGC3 and show abundant expression at the mRNA and protein level, while the expression is suppressed in hypermethylated carcinoma cell lines (R=-0.73). Short-interfering RNA-mediated reduction of PCDHGC3 led to a decrease of apoptosis in RG/C2 adenoma cells, and overexpression of PCDHGC3 in HCT116 cells resulted in the reduction of colony formation, consistent with tumour suppressor capabilities for PCDHGC3. Further functional analysis showed that PCDHGC3 can suppress Wnt and mammalian target of rapamycin signalling in colorectal cancer cell lines. Taken together, our data suggest that the PCDH LRES is an important tumour suppressor locus in colorectal cancer, and that PCDHGC3 may be a strong marker and driver for the adenoma-carcinoma transition.

Repression of KIAA1199 attenuates Wnt-signalling and decreases the proliferation of colon cancer cells.

BACKGROUND: The KIAA1199 transcript is upregulated in colon adenomas and downregulated upon ß-catenin knockdown. METHODS: Transcript profiling was performed on >500 colon biopsies, methylation profiling data were compared with transcript data. Immunohistochemistry assessed KIAA1199 protein expression in 270 stage II/III tumours (>3 years follow-up). The effects of stable KIAA1199 knockdown in SW480 cells (three different constructs) were studied using transcriptional profiling, proliferation and protein analysis. RESULTS: The KIAA1199 transcript was strongly upregulated in 95% of adenocarcinomas. Absent expression in normal mucosa correlated with KIAA1199 promotor methylation. Nuclear and cytoplasmic KIAA1199 protein expression was identified in colon adenocarcinomas and other types of cancers. A subpopulation of patients with tumours strongly expressing KIAA1199 in the nucleus showed a better outcome with regard to recurrence as lung or liver metastases. The KIAA1199 knockdown affected the cell cycle and the Wnt-signalling pathway. Reduced cellular proliferation and decreased KI67, phosphorylated retinoblastoma, ß-catenin and ASCL2 protein expression supported these findings. Eighteen Wnt-signalling genes differentially expressed upon KIAA1199 knockdown correlated with the KIAA1199 expression profile in clinical specimens. CONCLUSION: The KIAA1199 knockdown attenuates the effects of the Wnt/ß-catenin signalling and it may thus be regarded as a regulatory part of this pathway.

Human herpesvirus 6B induces phenotypic maturation without IL-10 and IL-12p70 production in dendritic cells.

Human herpesvirus 6B (HHV-6B) is the causative agent of the common childhood febrile illness, exanthema subitum. The virus is predominantly regarded as a T-cell tropic virus, although in reality it has the ability to infect a wide variety of cell types including monocytes, macrophages and dendritic cells (DC). Although DC are important immune regulators, the modulating effects of HHV-6B on DC are controversial. Here, we examine the phenotypic and functional consequences of HHV-6B infection of DC. The addition of HHV-6B to immature DC led to expression of the nuclear viral p41 protein and cell surface expression of the viral glycoprotein gp60/110 consistent with HHV-6B infection. Nevertheless, HHV-6B did not induce noticeable cytopathogenic effects or cell death in infected DC. Importantly, HHV-6B infection induced a partial phenotypic maturation of immature DC as demonstrated by a substantial increase in the expression of HLA-DR, CD86 and CD40, whereas only a minor increase in CD80 and CD83 was observed. This phenotypic maturation was, however, not followed by functional maturation, because HHV-6B infection did not induce IL-10 and IL-12p70 production in immature DC. However, infected DC were still able to react to bacteria-derived stimuli such as lipopolysaccaharide by an even more pronounced production of IL-10 and IL-12p70 when compared to that of uninfected DC.

[Seroprevalence of Lyme Borreliosis and tick-borne encephalitis in workers at risk, in eastern France]. / Séroprévalence de la borréliose de Lyme et de l'encéphalite à tiques chez des professionnels exposés dans le Grand est de la France.

OBJECTIVE: The aim of this article was to assess the seroprevalence of Lyme Borreliosis and tick-borne encephalitis (TBE) among occupationally exposed forest workers. METHODS: Workers exposed to tick bites in Eastern France were interviewed by occupational health physicians of the mutualité sociale agricole (MSA) on their sociodemographic features, their occupational activity, their last tick bite, their clinical history, and their means of prevention. Blood sampling was carried out for antibody detection. RESULTS: Among the 2975 subjects included in the study, the observed seroprevalence was 14.1% for Lyme borreliosis and 3.4% for TBE. Age, occupational activity, and place of residence significantly influenced the serological status of Lyme borreliosis. The seroprevalence was significantly higher among woodcutters (17.5%) than among other occupational categories (p<.001). Seroprevalence in Alsace (26.9%) and Lorraine (16.5%) were significantly higher than in other regions (p<0.001 and p<0.01, respectively). The seroprevalence of TBE was significantly higher in Alsace (5.5%; p<0.001). The rates of seroprevalence for both infections varied according to forest areas. The multifactorial analysis of prevention practices revealed three types of behaviors as far as protection was concerned: "rigorous", "partial", or "insufficient". CONCLUSION: These results do not change the present French indications for use of TBE vaccine. They highlight the importance of information on these diseases and the need for further studies on microbial ecology and risk-factors identification.

Human herpesvirus 6B induces phosphorylation of p53 in its regulatory domain by a CK2- and p38-independent pathway.

Here, we demonstrate that human herpesvirus 6B (HHV-6B) infection upregulates the tumour suppressor p53 and induces phosphorylation of p53 at Ser392. Interestingly, phosphorylation at the equivalent site has previously been shown to correlate with p53 tumour suppression in murine models. Although the signalling pathways leading to Ser392 phosphorylation are poorly understood, they seem to include casein kinase 2 (CK2), double-stranded RNA-activated protein kinase (PKR), p38 or cyclin-dependent kinase 9 (Cdk9). By using column chromatography and in vitro kinase assays, CK2 and p38, but not PKR or Cdk9, eluted in column fractions that phosphorylated p53 at Ser392. However, treatment of cells with neither the CK2 and Cdk9 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) nor p38 kinase inhibitors reduced HHV-6B-induced Ser392 phosphorylation significantly. Knockdown of the CK2beta subunit or p38alpha by small interfering RNA had no effect on HHV-6B-induced phosphorylation of p53 at Ser392. Thus, HHV-6B induces p53 Ser392 phosphorylation by an atypical pathway independent of CK2 and p38 kinases, whereas mitogen-activated protein (MAP) kinase signalling pathways are involved in viral replication.

Aluminum concentration in deciduous teeth is dependent on tooth type and dental status.

Aluminum (Al) concentration was assessed in deciduous teeth in relation to sex, year of birth, tooth type, and the presence of caries and roots. Three hundred and twenty-three deciduous teeth from children born during the period 1952 93 in a county in southeast Sweden were sampled, and the Al content determined by graphite furnace atomic absorption spectrophotometry. The arithmetic mean of the Al concentration was 0.58 +/- 0.64 ppm dry weight (mean +/- standard deviation) and differed significantly between incisors (1.05 +/- 1.04 ppm) and canines (0.48 +/- 0.50 ppm) and between incisors and molars (0.53 +/- 0.55 ppm). A significant difference was found between teeth with and without caries. No significant differences were found between sexes. The Al concentration correlated significantly with tooth weight for incisors (r = -0.47) and canines (r = -0.45) but not for molars (r = 0.03). No significant change in Al concentration was found over time. Caries-free deciduous molars are suggested as the most useful teeth for biological monitoring of aluminum.

Polyclonal antibodies against NCAM and tenascin delay endplate reinnervation.

Experiments were performed to block molecules with antibodies which are upregulated in nerve and muscle following denervation. The delay in endplate reinnervation was taken as a measure for their involvement in regeneration. Gluteus maximus muscles of 86 male CBA/J mice were hemidenervated by freezing the caudal gluteal nerve at a defined position. The degree of reinnervation was evaluated in identified endplates by repeated vital staining of ACh receptors with rhodaminated alpha-bungarotoxin and of axons with 4Di-2ASP. Normally, endplates were completely reinnervated by 13-14 days (108 endplates in seven muscles). After daily application of polyclonal antibodies against NCAM or tenascin, reinnervation was significantly delayed. Preimmune serum, rabbit immunoglobulins or saline did not show this effect. Several monoclonal antibodies against NCAM (H-28) and tenascin (576, 578, 630, 633) showed a tendency but no significant effect. It is concluded that both NCAM and tenascin, upregulated after denervation, are involved in axon guidance and/or endplate reinnervation.

Regular and photodamage-enhanced remodelling in vitally stained frog and mouse neuromuscular junctions.

Repeated in vivo observations of vitally stained neuromuscular junctions allow direct monitoring of ongoing structural changes, although, normally occurring changes (remodelling) and those inflicted by the illumination itself (photodamage) need to be dissociated. In frog cutaneus pectoris muscles, stained in vivo with 4Di-2ASP twice within four to five weeks, growth only was observed in 14 out of 92 junctions (in 18 muscles), retraction only in 19, and both features simultaneously in 22 junctions, while 37 junctions showed no changes. The summed growth in a junction amounted to 5-42 microns, retraction to 5-52 microns, while overall changes in synaptic length were absent. This and the simultaneous occurrence of both, growth and retraction within a single junction, indicates junctional remodelling. Similar amounts of remodelling were observed in junctions illuminated for 180, 60 or 10-30 s, indicating that within this range and with the given optical system, blue light and 4Di-2ASP fluorescence were not harmful. Remodelling was not induced by the experimental procedure per se, since in in vitro preparations signs of sprouting (and retraction) were equally frequent in junctions of totally untreated muscles, in junctions vitally stained four to five weeks previously or vitally stained but not light-exposed in the same muscle. In endplates of mouse gluteus maximus muscles double stained with 4Di-2ASP and rhodamine-alpha-bungarotoxin, unusually large (> 10 microns) terminal sprouts and retraction with gain loss of ACh-receptors became prominent 9-29 days following illumination for more than 60 s. Frequently also, muscle fibre damage with local contracture and acute loss of stainability of axon terminals occurred; often followed by muscle fibre denervation. In contrast, no such changes occurred after lower illumination intensities (12% emission filter for green) or shorter exposure times (< 60 s); even at a fourth exposure performed within 53 days. Previously reported smaller changes indicating regular remodelling were not investigated here.

Evaluation of cause-related periodontal therapy and compliance with maintenance care recommendations.

An evaluation of the long-term clinical effects of an intense period of cause-related periodontal therapy provided by dental hygiene students, was made in patients with moderately advanced periodontitis. By the evaluation, we also intended to gain information about compliance with given recommendations for periodontal health maintenance. The results after 3 years without supervision by the specialist team showed that achieved beneficial effects on the gingival conditions were maintained despite a significant increase in plaque prevalence. Recommendations as to the daily use of a variety of additional oral hygienic measures besides toothbrushing met with a considerable lack of compliance. Maintenance visits to the referring general practitioner were mostly made once a year and included regular dental care. Despite this, no further deterioration of periodontal status was observed. The results indicate that it may be possible to maintain successful effects of periodontal therapy in this patient category with less personal and professional effort than traditionally recommended.