Methylation profiles of hereditary and sporadic ovarian cancer

Bol G M, Suijkerbuijk K P M, Bart J, Vooijs M, van der Wall E & van Diest P J
(2010) Histopathology 57, 363–370
Methylation profiles of hereditary and sporadic ovarian cancer Aims: Tumour suppressor gene silencing through promoter hypermethylation plays an important role in oncogenesis. Carcinogenesis of hereditary cancers usually differs from that of their sporadic counterparts, but methylation has hardly been studied in hereditary ovarian cancer. The aim of this study was to investigate promoter methylation of a set of common tumour suppressor genes in BRCA1‐related ovarian cancer in comparison with sporadic ovarian cancer. Methods and results: Methylation‐specific multiplex ligation‐dependent probe amplification was used to assess the extent of promoter methylation of 24 different tumour suppressor genes in BRCA1‐associated (n = 25) and matched sporadic ovarian tumours (n = 50). A cumulative methylation index (CMI) was calculated and differences between individual genes were analysed. There was no significant difference in cumulative methylation between BRCA1‐associated and sporadic ovarian carcinomas (median CMI 108; CMI 110; P = 0.86). Also, methylation patterns of individual genes did not show distinct differences after correction for multiple comparisons. CDH13, GSTP1 and RASSF1 were frequently methylated in both sporadic and hereditary ovarian cancers. BRCA1 methylation occurred in 14% of sporadic tumours, but was not detected in BRCA1‐associated tumours. Conclusions: CDH13, GSTP1 and RASSF1 are frequently methylated in both sporadic and BRCA1‐associated ovarian cancers. Interestingly, methylation of BRCA1, while frequent in sporadic ovarian cancer, never occurred in the hereditary group. BRCA1‐associated ovarian cancers mimic their sporadic counterparts in extent and pattern of promoter methylation of several common tumour suppressor genes. This finding could have implications for future chemotherapy regimens based on epigenetic changes.     

Retention in substance dependence treatment: the relevance of in-treatment factors

In addition to “fixed” patient demographic and background variables, treatment process constructs play an important role in the prediction of treatment retention in substance dependence treatment. The objective of this paper is to analyze the predictive role of repeated measures of treatment readiness and behavioral intention, and of patients' perception of the therapeutic alliance, while controlling for fixed patient-oriented variables. Ninety-three patients, both alcohol and drug dependents, enrolled in this study, which was conducted in an inpatient treatment setting. Patients completed questionnaires shortly after admission (t=0) and approximately 2 weeks later (t=1). Using these measures, 35% of variance of a length of stay in treatment of up to 30 days could be explained. Fixed patient-oriented variables accounted for 21% of variance. Of the cognitive factors, helping alliance was the most important, accounting for an additional 8% of variance. The implications of these results are discussed.     

Armed against falls: the contribution of arm movements to balance recovery after tripping

Arm movements after perturbations like tripping over an obstacle have been suggested to be aspecific startle responses, serve a protective function or contribute to balance recovery. This study aimed at determining if and how arm movements play a functional role in balance recovery after a perturbation. We tripped young subjects using an obstacle that suddenly appeared from the floor at exactly mid-swing. We measured arm muscle EMG, quantified body rotations after tripping, and established the effects of arm movements by calculating how the body would have rotated without arms. Strong asymmetric shoulder muscle responses were observed within 100 ms after trip initiation. Significantly faster and larger responses were found in the contralateral arm abductors on the non-tripped (right) side. Mean amplitudes were larger in the ipsilateral retroflexors and contralateral anteflexors. The resulting asymmetric arm movements had a small effect on body rotation in the sagittal and frontal planes, but substantially affected the body orientation in the transverse plane. With the enlargement of the ongoing arm swing, the arms contributed to balance recovery by postponing the transfer of arm angular momentum to the trunk. This resulted in an axial rotation of the lower segments of the body towards the non-tripped side, which increases the length of the recovery step in the sagittal plane, and therefore facilitates braking the impending fall.     

The Accurate Control of Ultrafiltration

An apparatus has been constructed to regulate ultra-filtration accurately during dialysis. The principle of the apparatus is that, per unit of time, exactly the same amount of dialysate is introduced into the dialyzer as is discharged from it. The apparatus consists of two isovolumetric pumps connected in line. The four compartments of the two pumps must change their functions at every pump stroke. This is accomplished by a switching system. There is a continuously closed dialysate circuit. The fluid extracted from this circuit will be replenished from the blood compartment of the dialyzer. Ultrafiltration is regulated by a simple peristaltic pump, which sucks the fluid out of the closed dialysate circuit. The isovolumetric pumps and the switching system are driven by the elevated pressure of the dialysate (0.5–1.0 atm). The apparatus can be used in single pass dialysis. Dialysis in accordance with the Bergström principle can be simply performed.
In over 5,000 dialyses with several types of dialyzers, ultrafiltration was always accurate within the measuring limits. Considerable improvement was noticed in the well-being of the patients; hypotension, nausea, vomiting and muscle cramps were not seen.     

Pathogenesis of Influenza A/H5N1 virus infection in ferrets differs between intranasal and intratracheal routes of inoculation

Most patients infected with highly pathogenic avian influenza A/H5N1 virus develop severe pneumonia resulting in acute respiratory distress syndrome, with extrarespiratory disease as an uncommon complication. Intranasal inoculation of ferrets with influenza A/H5N1 virus causes lesions in both the respiratory tract and extrarespiratory organs (primarily brain). However, the route of spread to extrarespiratory organs and the relative contribution of extrarespiratory disease to pathogenicity are largely unknown. In the present study, we characterized lesions in the respiratory tract and central nervous system (CNS) of ferrets (n = 8) inoculated intranasally with influenza virus A/Indonesia/5/2005 (H5N1). By 7 days after inoculation, only 3 of 8 ferrets had a mild or moderate bronchointerstitial pneumonia. In contrast, all 8 ferrets had moderate or severe CNS lesions, characterized by meningoencephalitis, choroiditis, and ependymitis, and centered on tissues adjoining the cerebrospinal fluid. These findings indicate that influenza A/H5N1 virus spread directly from nasal cavity to brain, and that CNS lesions contributed more than pulmonary lesions to the pathogenicity of influenza A/H5N1 virus infection in ferrets. In comparison, intratracheal inoculation of ferrets with the same virus reproducibly caused severe bronchointerstitial pneumonia. The method of virus inoculation requires careful consideration in the design of ferret experiments as a model for influenza A/H5N1 in humans.     

Introduction of large DNA inserts into the barley pathogenic fungus, <Emphasis Type="Italic">Ustilago hordei</Emphasis>, via recombined binary BAC vectors and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

Genetic transformation of organisms with large genome fragments containing complete genes, with regulatory elements or clusters of genes, can contribute to the functional analysis of such genes. However, large inserts, such as those found on bacterial artificial chromosome (BAC) clones, are often not easy to transfer. We exploited an existing technique to convert BAC clones, containing genomic DNA fragments from the barley-covered smut fungus Ustilago hordei to binary BACs (BIBACs) to make them transferable by the Agrobacterium tumefaciens T-DNA transfer machinery. Genetic transformation of U. hordei with BAC clones using polyethylene glycol or electroporation is difficult. As a proof of concept, two BAC clones were successfully converted into BIBAC vectors and transferred by A. tumefaciens into U. hordei and U. maydis, the related corn smut fungi. Molecular analysis of the transformants showed that the T-DNA containing the BAC clones with their inserts was stably integrated into the U. hordei genome. A transformation frequency of approximately 10⁻⁴ was achieved both for U. hordei sporidia and protoplasts; the efficiencies were 25–30 times higher for U. maydis. The combination of in vivo recombineering technology for BAC clones and A. tumefaciens-mediated transformation of Ustilago species should pave the way for functional genomics studies.     

Evaluation of a new multiplex polymerase chain reaction assay STDFinder for the simultaneous detection of 7 sexually transmitted disease pathogens

We evaluated a new multiplex polymerase chain reaction (mPCR), “STDFinder assay”, a novel multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous detection of 7 clinically relevant pathogens of STDs, i.e., Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium, Treponema pallidum, and herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). An internal amplification control was included in the mPCR reaction. The limits of detection for the STDFinder assay varied among the 7 target organisms from 1 to 20 copies per MLPA assay. There were no cross-reactions among any of the probes. Two hundred and forty-two vaginal swabs and an additional 80 specimens with known results for N. gonorrhoeae and C. trachomatis, obtained from infertile women seen at an infertility research clinic at the Kigali Teaching Hospital in Rwanda, were tested by STDFinder assay and the results were confirmed by single real-time PCR using different species-specific targets. Compared to the reference standard, the STDFinder assay showed specificities and sensitivities of 100% and 100%, respectively, for N. gonorrhoeae, C. trachomatis, and M. genitalium; 90.2% and 100%, respectively, for Trichomonas vaginalis; and 96.1% and 100%, respectively, for HSV-2. No specimen was found to be positive for HSV-1 by either the STDFinder assay or the comparator method. Similarly, the sensitivity for Treponema pallidum could not be calculated due to the absence of any Treponema pallidum-positive samples. In conclusion, the STDFinder assays have comparable clinical sensitivity to the conventional mono and duplex real-time PCR assay and are suitable for the routine detection of a broad spectrum of these STDs at relatively low cost due to multiplexing.     

A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth

The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies have been successfully used, such as cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities. We have previously reported on the successful isolation of antagonistic anti-EGFR nanobodies. In our study, we aimed to improve the efficacy of these molecules by combining nanobodies with specificities similar to both cetuximab and matuzumab into a single biparatopic molecule. Carefully designed phage nanobody selections resulted in two sets of nanobodies that specifically blocked the binding of either matuzumab or cetuximab to EGFR and that did not compete for each others' binding. A combination of nanobodies from both epitope groups into the biparatopic nanobody CONAN-1 was shown to block EGFR activation more efficiently than monovalent or bivalent (monospecific) nanobodies. In addition, this biparatopic nanobody potently inhibited EGF-dependent cell proliferation. Importantly, in an in vivo model of athymic mice bearing A431 xenografts, CONAN-1 inhibited tumour outgrowth with an almost similar potency as the whole mAb cetuximab, despite the fact that CONAN-1 is devoid of an Fc portion that could mediate immune effector functions. Compared to therapy using bivalent, monospecific nanobodies, CONAN-1 was clearly more potent in tumour growth inhibition. These results show that the rational design of biparatopic nanobody-based anticancer therapeutics may yield potent lead molecules for further development.     

Different stages of intraplaque hemorrhage are associated with different plaque phenotypes: a large histopathological study in 794 carotid and 276 femoral endarterectomy specimens

Background and purpose

Intraplaque hemorrhage (IPH) is an important determinant of progression and destabilization of atherosclerotic plaque. We recently demonstrated that IPH is an independent predictor of cardiovascular events. IPH has become more clinically relevant since magnetic resonance imaging (MRI) technique is able to visualize IPH in vivo. Different stages of IPH have been described. However, etiology of the different stages is not known and it is unclear if these detected different stages are all associated with the vulnerable plaque phenotype.

Methods and results

1070 patients who underwent a carotid (n = 794) or femoral (n = 276) endarterectomy were included. Histopathological presence of IPH was determined and divided into 3 types: recent, organized and amorphous IPH. Carotid IPH was observed in 644/794 (81%) plaques, divided into 14 (2%) recent, 70 (11%) organized and 560 (87%) amorphous. Femoral IPH was observed in 175/276 (63%) plaques, divided into 2 (1%) recent, 89 (51%) organized and 84 amorphous (48%). Overall presence of carotid IPH was associated with a large lipid core, no or minor staining of smooth muscle cells, no or minor calcification and high microvessel density. Overall presence of femoral IPH was associated with moderate to heavy staining of macrophages. Plaques with organized IPHs revealed more macrophages, a larger lipid core, less smooth muscle cells, less calcification and higher microvessel density than plaques with amorphous IPHs.

Conclusions

IPH is a significant characteristic of carotid and femoral atherosclerotic plaque and can be classified into different types. Organized IPH is associated with unstable and amorphous IPH with stable plaque characteristics.